Infectivity-enhanced conditionally-replicative adenovirus and uses thereof

ABSTRACT

A modified adenovirus capable of overcoming the problem of low level of coxsackie-adenovirus receptor (CAR) expression on tumor cells and methods of using such adenovirus are provided. The fiber protein of the adenovirus is modified by insertion or replacement so as to target the adenovirus to tumor cells, and the replication of the modified adenovirus is limited to tumor cells due to specific promoter control or mutations in E1a or E1b genes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of prior application U.S. Ser. No.10/697,535, filed on Aug. 20, 2003, now abandoned, which claims thebenefit of priority to U.S. Ser. No. 09/569,789, filed May 12, 2000, nowU.S. Pat. No. 6,824,2004, which claims benefit of provisional patentapplication Ser. No. 60/133,634, filed May 12, 1999, now abandoned;wherein each application is hereby incorporated by reference in itsentirety.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through a grantfrom the National Institutes of Health. Consequently, the federalgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to adenoviral vectors. Morespecifically, the present invention relates to infectivity-enhancedconditionally replicative adenovirus vectors.

2. Description of the Related Art

Surgery, chemotherapy and radiotherapy constitute the conventionaltherapies in clinical use to treat cancer. These therapies have produceda high rate of cure in early-stage cancer, but most late-stage cancersremain incurable because they cannot be resected or the dose ofradiation or chemotherapy administered is limited by toxicity to normaltissues. An alternative promising approach is the transfer of geneticmaterial to tumor or normal cells as a new therapy itself or to increasethe therapeutic index of the existing conventional therapies. In thisregard, three main strategies have been developed to accomplish cancergene therapy: potentiating immune responses against tumors, elicitingdirect toxicity to tumors, and compensating the molecular lesions oftumor cells.

To achieve the high level of gene transfer required in most cancer genetherapy applications, several viral and non-viral vectors have beendesigned. Adenoviral vectors have been used preferentially over otherviral and non-viral vectors for several reasons, including infectivityof epithelial cells, high titers, in vivo stability, high levels ofexpression of the transgene, gene-carrying capability, expression innon-dividing cells, and lack of integration of the virus into thegenome. In most of the adenoviral vectors used in cancer gene therapy,the transgene substitutes for the early 1 region (E1) of the virus. TheE1 region contains the adenoviral genes expressed first in theinfectious stage and controls expression of the other viral genes. Theearly region 3 (E3) gene codes for proteins that block a host's immuneresponse to viral-infected cells and is also usually deleted in vectorsused for cancer gene therapy, particularly in immunopotentiatingstrategies.

E1-substituted, E3-deleted vectors can carry up to 8 kb of non-viralDNA, which is sufficient for most gene therapy applications.E1-substituted, E3-deleted vectors are propagated in packaging celllines that transcomplement their E1-defectiveness, with productionyields of up to 10,000 virion particles per infected cell, dependingupon the transgene and its level of expression in the packaging cell.Not all of the viral particles are able to transduce cells or toreplicate in the packaging cell line, so bioactivity of a particularvector has been defined as the ratio of functional particles to totalparticles. This bioactivity varies from 1/10 to 1/1000, depending notonly upon the vector, but also upon the methods of purification andquantification. The titer used is the concentration of functionalparticles, which can be as high as 10¹² per milliliter.

One problem encountered when propagating these vectors to high titers isthe recombination of vector sequences with the E1 sequences present inthe packaging cell line, thereby producing replication-competentadenoviruses (RCA). This problem has been solved by using packaging celllines where the E1 gene does not overlap with the vector sequences.

The current generation of adenoviral vectors are limited in their usefor cancer gene therapy, primarily for three reasons: (1) the vectorsare cleared by the reticuloendothelial system; (2) the vectors areimmunogenic; and (3) the vectors infect normal cells. The problem offiltration by the reticuloendothelial system cells such as macrophagesof the spleen or Kupffer cells of the liver affects adenoviral vectorsas well as other viral and non-viral vectors and limits their utility inintravascular administration. The early and late viral genes that remainin E1-E3 deleted vectors may also be expressed at low, but sufficientenough levels such that the transduced cells are recognized and lysed bythe activated cytotoxic T lymphocytes. Additionally, a higher viral dosemust be injected to reach the entire tumor before a neutralizing immuneresponse develops. The major limitation then becomes the amount ofvector that can be safely administered, which will depend upon thecapacity of the vector to affect tumor cells without affecting normalcells.

The limitations of adenoviral vectors at the level of infectivity istwo-fold. On the one hand, human clinical trials with adenoviral vectorshave demonstrated relatively inefficient gene transfer in vivo. This hasbeen related to the paucity of the primary adenovirus receptor,coxsackie-adenovirus receptor (CAR), on tumor cells relative to theircell line counterparts. On this basis, it has been proposed that genedelivery via CAR-independent pathways may be required to circumvent thiskey aspect of tumor biology. On the other hand, adenoviral vectorsefficiently infect normal cells of many epithelia. This results in theexpression of the transgene in normal tissue cells with the consequentadverse effects. This problem has been addressed by targeting adenoviralvectors to tumor cells at the level of receptor interaction andtransgene transcription.

Targeting adenoviral vectors to new receptors has been achieved by usingconjugates of antibodies and ligands, in which the antibody portion ofthe conjugate blocks the interaction of the fiber with the CAR receptorand the ligand portion provides binding for a novel receptor. Receptortargeting has also been achieved by genetic modification of the fiber.

Transcriptional targeting of adenoviral vectors has been demonstratedusing tumor-antigen promoters or tissue-specific promoters to controlthe expression of the transgene. However, these promoters can lose theirspecificity when inserted in the viral genome and, depending upon thelevel of toxicity of the transgene, even low levels of expression can bedetrimental to normal cells. Thus, for cancer gene therapy, the majorissues limiting the utility of adenoviral vectors are the efficiency andspecificity of the transduction.

A major limitation found in the use of adenoviral vectors in theclinical setting is the number of tumor cells that remain unaffected bythe transgene. A vector that propagates specifically in tumor cells,results in lysis and subsequently allows for transduction of neighborcells by newly produced virions will increase the number of tumor cellsaffected by the transgene. A good replicative vector should be weaklypathogenic or non-pathogenic to humans and should be tumor-selective.Efforts have been aimed at improving the safety of replication-competentadenoviruses with the goal of being able to administer much higherdoses. One strategy is to transcomplement the E1 defect with anE1-expression plasmid conjugated into the vector capsid, which allows asingle round of replication thereby producing a new E1-substitutedvector with the ability of local amplification and subsequent genetransduction.

Other strategies are designed to obtain vectors that replicatecontinuously and whose progeny are also able to replicate, but areincapable of propagating in normal cells. In this regard, two approacheshave been described that render adenovirus propagation selective fortumor cells: (1) deletions, and (2) promoter regulation. Adenoviralmutants unable to inactivate p53 propagate poorly in cells expressingp53 but efficiently in tumor cells where p53 is already inactive. Basedupon this strategy, an adenovirus mutant in which the E1b-55k viralprotein was deleted and was unable to bind to p53 was effective ineliminating tumors in preclinical models and is in clinical trials.Controlling viral replication by substituting a viral promoter, such asthe E1a promoter, with a tumor associated-antigen promoter, such as thealpha-fetoprotein promoter or the prostate antigen promoter, has beendemonstrated, and specific lysis of tumors transfected with anadenovirus vector expressing either of the abovementioned promoters wasdemonstrated in murine models.

Both approaches have limitations, however. The fact that other viralproteins besides E1b-55K also interact with p53, and because p53 can benecessary for the active release of virus in the later stages ofinfection may affect the specificity of the vector. Another caveatresults from using E1a as the only controlled viral gene since E1a-likeactivity has been found in many tumor cell lines. Furthermore, theactual specificity of the above-mentioned promoters for cancer cells,and the fact that promoters inserted in the viral genome can lose theirexpression specificity are factors that hindered clinical applicationsof this approach.

Therefore, new methods are clearly needed to achieve more selectivetherapeutic effects of replication-competent adenoviruses. For thesevectors, in parallel to what has been achieved with non-replicativevectors, modification of viral tropism could enhance tumor transductionand tumor selectivity at the level of cell entry, and in this wayrealize the full potential of replicative vectors for cancer genetherapy.

The prior art is deficient in adenoviral vectors that are specific for aparticular cell type (i.e., do not infect other cell types) and thatreplicate with high efficiency in only those particular cell types. Thepresent invention fulfills this long-standing need in the art.

SUMMARY OF THE INVENTION

Adenoviral vectors have been widely employed in cancer gene therapy.Their high titers, structural stability, broad infectivity, high levelsof transgene expression, and lack of integration have contributed to theutility of this vector. In this regard, adenoviral vectors have beenused to transfer a variety of genes such as cytokines, tumor suppressergenes, pro-drug converting genes, antisense RNAs and ribozymes toinhibit the expression of oncogenes, antiangiogenic genes, etc. Despitethe promise of adenoviral vectors, results from experimental models andclinical trials have been less than optimal.

Within this context, several specific limitations have been identified.One limitation lies in the poor infectability of primary tumors due tolow levels of the primary adenovirus receptor CAR. A second limitationthat particularly affects the efficiency of replicative vectors isrelated to the lack of tumor-specific replication achieved usingpromoters or mutations. The present invention describes methods toincrease adenovirus infectivity based upon modification of the virustropism. The present invention demonstrates that modification of theadenovirus fiber by genetic manipulation increases infectivity ofprimary tumors several orders of magnitude due to CAR-independent genetransfer. In addition, selective replication in tumors is describedherein, and represents a safe and effective means to lyse and transducetumors. The present invention further describes a strategy based uponcontrol of the expression of one or more essential early viral genesusing tumor-specific promoters.

It is a goal of the present invention to improve the infectivity andspecificity of conditional replicative vectors, thereby improving theirtherapeutic utility and efficacy.

One object of the present invention is to provide adenoviral vectorsthat possess enhanced infectivity to a specific cell type (i.e., thatare not limited to CAR-dependent cell entry) and that replicate withhigh efficiency in only those cell types.

In one embodiment of the present invention, there is provided aninfectivity-enhanced conditionally-replicative adenovirus. Thisadenovirus possesses enhanced infectivity towards a specific cell type,which is accomplished by a modification or replacement of the fiber ofthe adenovirus. The modification is accomplished by introducing a fiberknob domain from a different subtype of adenovirus, introducing a ligandinto the HI loop of the fiber knob, or replacing the fiber with asubstitute protein which presents a targeting ligand. Additionally, theadenovirus has at least one conditionally regulated early gene, suchthat replication of the adenovirus is limited to the specific cell type.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that an anti-knob Fab-FGF2 conjugate enhances celltransduction. FIG. 1A shows that AdCMVluc (5×10⁷ pfu) was preincubatedwith 1.44 μg of Fab or 1.94 μg of Fab-FGF2. SKOV3 cells (24,000 cellsper well in 24-well plates) were infected with control vector or withthe vector complexes (MOI of 50) Inhibition was performed by adding apolyclonal anti-FGF2 to the complex before infection. Luciferaseactivity in cell lysates was assayed 24 h after infection. The mean oftriplicate experiments is shown. FIG. 1B shows that AdCMVLacZ wascomplexed with Fab-FGF2 conjugate as in FIG. 1A. SKOV3 cell wereinfected with control vector (a, c) or complexed vector (b, d) at MOI of5 (a, b) or 50 (c, d) and stained with X-gal 24 h after infection.

FIG. 2 shows that Fab-FGF2 retargeting augments in vivo therapeuticbenefit of the AdCMVHSV-TK vector. Five days after i.p. inoculation of2×10⁷ SKOV3 cell in SCID mice, 2×10⁸ or 2×10⁹ pfu of AdCMVTK alone orcomplexed with FGF2 were injected i.p. Forty eight h later, half ofthe-mice were treated with GCV (50 mg/kg body weight) for 14 days.Survival was monitored daily.

FIG. 3 shows the HI loop of the fiber as a domain to insert ligand forretargeting adenoviruses. FIG. 3A shows the knob trimer viewed along thethree-fold symmetry axis (Reproduced from Xia et al. [42]). FIG. 3Bshows the localization of targeting ligands within the fiber molecule.

FIG. 4 shows a comparison of the gene transfer efficiencies to culturedovarian cancer cells mediated by AdCMVLuc and Ad5IucRGD. Human ovariancancer cells SKOV3.ipl (FIG. 4A) and OV-4 (FIG. 4B) were transduced withAdCMVLuc or Ad5lucRGD at an MOI of 1 or 10 pfu/cell essentially asdescribed in for 293, HUVEC and RD cells. Recombinant Ad5 fiber knobprotein was added to cells prior to infection with the virus. Each datapoint is the average of three independent measurements obtained in oneexperiment.

FIG. 5 shows transduction of primary cells isolated from ascitesobtained from ovarian cancer patients. Cells isolated from ascites oftwo (FIGS. 5A and B) ovarian cancer patients were transduced withAdCMVLuc or Ad5lucRGD at MOI of 1 or 10 in the presence or absence ofblocking Ad5 fiber knob protein. The data points represent the mean ofthree independent determinations.

FIG. 6 shows enhancement of adenovirus infectivity by RGD modificationof the fiber knob. Triplicates of A549 cells (panel A) and LNCaP cells(panel B) were transduced with increasing doses of either Ad5luc orAd5lucRGD. After 36 h, cell transduction was determined by luciferaseassay. The data are presented as relative light units (RLU) normalizedto mg of cellular protein. The results show an infectivity advantage ofthe RGD modified vector over the non-modified one in both cell lines.

FIG. 7 shows a conceptual representation of the conditional replicationenablement system for adenovirus. The initial introduction ofrecombinant virus into the tumor mass infects the cells shown ascircles. The replication enabling plasmid converts these cells invector-producing cells. The produced vector can infect adjacent cells(arrows).

FIG. 8 shows functional analysis of pE1FR. LS174T cells werecotransduced with the plasmid indicated in the abscissa as a liposomiccomplex (0.5 μg DNA/1.0 μg DOTAP:DOPE) and AdCMVluc (MOI=1). Forty eighthours after transduction, the amount of virus present in the lysate ofcells was measured by a plaque assay in 293 cells.

FIG. 9 shows enhancement of E1-defective adenoviral transgene expressionby pE1FR administration. Nude mice engrafted with human lungadenocarcinoma tumors (A549 cell line) received an intratumoralinjection of E1-defective virus AdCMVluc (10⁸ pfu per 8-10 mm diametertumor) mixed with plasmid pE1FR or pUC13 (3 μg). One week later,luciferase expression in tumors was measured. Each bar represents onemouse with a pair of tumors, one treated with AdCMVluc and pE1FR and theother one with AdCMVluc and pUC13. The ratio of luciferase expression inthe tumor treated with pEIFR versus the one treated with pUCI3 is shown.

FIG. 10 shows the E1A-like activity of IL-6 can be exploited to produceAd312 virions in HepG2 cells and in a variety of cell lines responsibleto IL-6. Cells (1 to 4×10⁵) were infected with wild type adenovirus orAd5dl312 at an MOI of 10 in the absence or presence of 100 units/ml ofrhIL-6. Six days later, cells were lysed and the amount of virus in thelysates was quantitated by plaque assay in 293 cells. For each cellline, bar from left to right represent wild type, wild type+IL-6, dl312,and dl312+IL-6.

FIG. 11 shows replication of Ad5dl312 and oncolytic effect in tumorcells without IL-6 addition. Ovarian carcinoma cells (OVCAR-3) wereinfected with E1-a deleted AD5dl312, wild type or E4-deleted AdSdl1014(MOI=10). FIG. 11 shows that six days post-infection, cells were lysedand the amount of virus in the lysates was measured by plaque assay in293 cells (for WT and dl312) or W162 cells (for dl1014).

FIG. 12 shows the analyses of adenoviral DNA. FIG. 12A shows the map ofE1A and fiber encoding regions of Ad5-D24RGD amplified by PCR, showingthe 24-bp deletion and the introduced RGD encoding sequence. FIG. 12Bshows restriction analysis of Ad5-D24RGD. The presence of the 24-bpdeletion was confirmed by BstX I digestion of the PCR product of the E1Aregion. The fragments were resolved on a 2% agarose gel, and visualizedby UV fluorescence. Marker: Gibco 1 Kb DNA ladder. The presence ofuncleaved PCR product verified the presence of the deletion (left). PCRamplification products of the region encoding the fiber from Ad5-D24 andAd5-D24RGD were resolved on a 6% acrylamide gel. Marker: Gibco 100 bpDNA ladder. The bigger size (27 bp) of Ad5-D24RGD band indicates thepresence of the sequence encoding RGD (right).

FIG. 13 shows propagation efficiency of Ad5-D24 versus Ad5-D24RGD. A549cells were infected with 0.01 particles/cell of Ad5-D24 or Ad5-D24RGDand incubated in medium containing 1 mCi/ml of BrdU. At the indicatedtimes after infection, the cells were harvested, and the encapsidatedDNA was purified by the spermine-Hcl method. Viral DNA from 6×10⁵infected cells was digested with HindIII, electrophoresed, and theresulting fragments were blotted into a membrane that was processed witha mouse anti-BrdU antibody. The amount of BrdU incorporated into viralDNA indicated that Ad5-D24RGD propagation is more efficient than that ofAd5-D24.

FIG. 14 shows oncolytic potency of the RGD-modified virus. FIG. 14 showscell viability analyzed with an XTT colorimetric assay. In both celllines, Ad5-D24RGD had higher lytic potency than did its unmodifiedcounterpart, as shown by the percentage of viable cells remaining in thecorresponding treatment conditions.

FIG. 15 shows in vivo oncolysis by high and low doses ofinfectivity-enhanced CRAds. FIG. 15A shows subcutaneous A549 xenograftsin nude mice treated with a single i.t. injection of 10⁹ viral particlesof Ad5lucRGD, Ad5-D24, Ad5-D24RGD, or with PBS alone. FIG. 15B showssubcutaneous A549 xenografts in nude mice treated with a single i.t.injection of 10⁷ viral particles of Ad5IucRGD, Ad5-D24, Ad5-D24RGD, orwith PBS alone. Tumor size was measured twice a week. Results are shownas fractional tumor volumes (V/V0, where V=volume at each time point;V0=volume at adenovirus injection), and each line represents the mean of5 tumors (±SD) in the high-dose group, and 4 tumors (±SD) in thelow-dose group. In the high-dose experiment, both CRAds show a similaroncolytic effect that results in smaller tumors compared to PBS treatedgroups (*Ad5-D24 p<0.05; **Ad5-D24RGD p<0.01). However, in the low-doseexperiment, tumors treated with Ad5-D24 followed a growth curve similarto that of tumors treated with non-replicative Ad5lucRGD; tumors treatedwith Ad5-D24RGD did not grow (p<0.01 compared to PBS). FIG. 15C showsthe detection of adenovirus hexon in tumor xenografts byimmunofluorescence. Frozen sections of tumor specimens injected with (a)Ad5lucRGD, (b) Ad5-D24, and (c) Ad5-D24RGD were treated with goatanti-hexon antibody and Alexa Fluo 488-labeled donkey anti-goatantibody, and nuclei were counterstained with Hoechst 33342. Images werecaptured from Leitz fluorescence microscope (100× magnification) with adouble filter. Sections taken from tumors treated with CRAds werepositive for adenovirus presence (green dots in b and c), beingAd5-D24RGD signal stronger than that of Ad5-D24. Samples taken fromtumors treated with PBS (not shown) or Ad5lucRGD exhibited no hexonsignal (a). i.t., intratumoral; vp, viral particles; Ad, adenovirus.

FIG. 16 shows in vivo oncolysis by systemic delivery ofinfectivity-enhanced CRAds. A total dose of 10⁹ viral particles dividedinto two consecutive doses of 5×10⁸/day of either Ad5lucRGD, Ad5-D24,Ad5-D24RGD, Ad5-wt, or PBS were injected in the tail vein of nude micebearing s.c. A549 xenografts. Tumor size was measured weekly. Resultsare shown as fractional tumor volumes (V/V0, where V=volume at each timepoint; V0=volume at adenovirus injection), and each line represents themean of 4 tumors (±SD). The data show that modification of the fiber tobroaden the tropism of a replicative adenovirus improves the oncolyticpotential in a systemic delivery context.

FIG. 17 shows schematic diagrams of Ad vectors containing VEGF promoter.These vectors are constructed from an E3 region-deleted Ad5 backbone anddo not contain the Ad E1A promoter region (from nucleotides 324 to 488of the Ad genome). Deletion of the E3 region was necessary due to thelength of the 2.6 kb VEGF. AdCMVE1 and AdVEGFE1 differ in the promoterdriving E1A expression.

FIG. 18A shows VEGF mRNA expression in various cell lines. The RT-PCRproduct for VEGF121(408 bp), VEGF165 (541 bp) or GAPDH (574 bp) is shownin upper or lower panel respectively. Lane 1, H82; lane 2, H460; lane 3,H157; lane 4, H322; lane 5, H522; lane 6, H1299; lane 7, QG56; lane 8,QG90; lane 9, A427; lane 10, H358; lane 11, A549; lane 12, N417 (lanes1-12 are lung cancer cell lines); lane 13, BEAS-2B, a normal bronchialepithelial cell line; lane 14, SKOV3.ipl, ovarian cancer cells; lane 15,MeWo, melanoma cells and lane 16, Panc-I, pancreatic cancer cells. FIG.18B shows VEGF protein expression in the same cell lines. 1×10⁵ cancercells were cultured for 24 h in the serum free media. The VEGF proteinconcentration in the media was measured by ELISA. Mean+SE of triplicatedetermination is shown.

FIG. 19 shows transgene expression by VEGF promoter in the Ad context invitro. Upper panel shows luciferase activities in various cell linesinfected by Ad5CMVLuc or Ad5VEGFLuc. 1×10⁵ cells of each cell line wereinfected with Ad5CMVLuc or Ad5VEGFLuc for 3 h at MOI 10. Cells wereharvested 48 h after infection and lysed in 100 ml of lysis buffer. Tenml of each lysate was used for luciferase assay. Mean+SE of triplicatedetermination is shown. Lower panel shows the ratio of VEGF promoteractivity to CMV promoter activity. To standardize the VEGF promoteractivity in each cell line, the luciferase activity with Ad5VEGFLuc wasexpressed as the percentage of luciferase activity with Ad5CMVLuc.

FIG. 20 shows tissue specificity of the VEGF promoter in the adenoviralcontext. Mice received 1×10⁹ pfu of Ad5VEGFLuc or Ad5CMVLuc via tailvein injection (three per group). Two days after virus injection, micewere sacrificed to obtain the organ samples. Each organ lysate wasassayed for lucifrase activity and normalized for protein concentration.Mean+SE of triplicate determination is shown.

FIG. 21 shows viral DNA replication 24 h after infection. 1×10⁵ cellswere infected with replication-competent Ads (Ad5VEGFE1, AdSCMVE1 or Ad5wt) or non-replicative Ad (Ad5VEGFLuc) at an MOI of 10 for 3 h and thencultured for 24 h. Viral DNA was isolated from the cells and analyzed byreal-time PCR analysis to evaluate adenoviral E4 copy number. E4 copynumbers were normalized by the b-actin DNA copy number. Mean+SE oftriplicate determination is shown.

FIG. 22A shows the cell killing effect of AdVEGFE1 evaluated by MTSassay. 5×10³ H157 cells were infected with Ad5CMVLuc (negative control),Ad5CMVE1 (positive control), or Ad5VEGFE1 at MOI of 0.1. After infectioncell viability in each well was quantified by MTS assay every threedays. The cell viability of cells infected with Ad5VEGFE1 or Ad5CMVE1 isexpressed as the percentage of the OD490 value to control cells infectedwith Ad5CMVLuc (100%). BEAS-2B cells were infected with each Ad at MOI10 and evaluated by MTS assay in the same manner. FIG. 22B shows thecell killing effect of AdVEGFE 1 evaluated by crystal violet staining2×10⁵ H157 cells and BEAS-2B cells were infected with each Ad at MOI0.1, 1.0 or 10. All wells were stained by crystal violet 9 days afterinfection to visualize viable cells.

FIG. 23 shows Ad5VEGFE1 suppressed tumor growth in vivo. Intact H157cells (5×10⁶) were injected s.c. into nude mice. When tumor formationwas seen 10 days after inoculation, 1×10⁸ pfu of each virus (diamond,Ad5CMVLuc; circle, Ad5VEGFE1; square, Ad5p53) was injected into thetumor directly. Three similar sized tumors were injected with eachvirus, and the mean volume+SE is shown.

FIG. 24 shows enhancement of infectivity to cancer cells with Ad5/3chimeric vector. 1×10⁵ cells of each cell line were infected byAd5CMVLuc or Ad5/3luc 1 at MOi 10. Infected cells were harvested 48 hafter infection and lysed in 100 ml of lysis buffer. Ten ml of eachlysate was used for luciferase assay. Mean+SE of triplicatedetermination is shown.

FIG. 25 shows enhancement of cell killing with Ad5/3 chimeric CRAd. Cellkilling effect was evaluated by MTS assay. 5×10³ cells of each cell linewere infected with Ad5CMVLuc (negative control), Ad5CMVE1 (positivecontrol), Ad5VEGFE1 or Ad5/3VEGFE1 at MOI of 1.0. After infection cellviability in each well was quantified using OD490 by MTS assay everythree days. The viability of cells infected with Ad5CMVE1, Ad5VEGFE1 orAd5/3VEGFE1 was expressed as a percentage of cells infected withAd5CMVLuc (100%).

FIG. 26 shows AdCK/CMV-Luc (black) demonstrates CAR-independent tropismversus Ad5/CMV-Luc (gray) in CAR-negative U118 cells and in the presenceof increasing free Ad5 knob in CAR-positive UI18-CAR cells. 100 viralparticles/cell, n=4, Bar=S.D.

FIG. 27 shows real-time PCR quantification of CXCR4 (gray) and Survivin(black) mRNA in various cell lines. Data expressed as copies/ng totalRNA.

FIG. 28 shows schematic of vectors Ad5/CMV-Luc, Ad5/CXCR4-Luc andAd5/Survivin-Luc. Promoter region is indicated for each.

FIG. 29 shows luciferase activities as percent of Ad5/CMV-Luc forAd5/CXCR4Luc and Ad5/Survivin-Luc at 48 h. 50 pfu/cell, n=3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses the two major limitations of replicativeadenoviral agents (viruses and vectors) in their application to cancergene therapy, i.e., the efficacy of transduction and the specificity ofreplication. Adenovirus binds to the coxsackie-adenovirus receptor, CAR,in the cellular membrane using the C-terminal globular domain of theviral fiber, the knob. Since a limited amount of coxsackie-adenovirusreceptor is present in tumors, one means to enhance infectivity would beto provide additional binding pathways. Therefore, two methods have beendeveloped to modify adenovirus binding. The first method uses a Fabfragment of an anti-knob antibody conjugated to a ligand of a cellularreceptor, while the second method comprises direct genetic modificationof the knob sequence.

One important advantage of direct genetic modification is that theprogeny will carry the modified fiber, thereby retaining the replicativevirus, enhanced infectivity trait through the amplification cycles.Wickman et al. (1997) have generated adenoviruses with chimeric fibersin which the ligand is connected to the carboxyl terminal position ofthe fiber. This carboxyl terminal location is not always appropriatebecause the addition of more than 20-30 heterologous amino acid residuescan result in the loss of fiber trimerization and binding to the capsid.Furthermore, the three-dimensional structure of the fiber indicates thatthe carboxyl terminal end points towards the virion, and therefore, awayfrom the cell surface. For these reasons, the HI loop was used herein asan exposed and amenable site for the incorporation of exogenoussequences.

It has been recognized that the major limitation in several strategiesof cancer gene therapy resides in the need to transduce the majority ofcells of a tumor. With the exception of a limited bystander effectdescribed in some strategies, the cells that are left untransduced willjeopardize and reduce any therapeutic effect. Adenoviral vectors arelimited in this regard by the paucity of its receptor, CAR, in tumors.It is a goal of the present invention to improve the infectivity ofadenoviral vectors by providing additional pathways to cell bindingbesides CAR. Previous data has shown that modification of the HI loop ofthe fiber is a feasible strategy to add new ligand motifs into thefiber. An RGD motif has already been incorporated into the fiber ofregular E1-deleted vectors and been shown to enhance the therapeuticeffects in vivo.

With regard to the efforts to increase the specificity at the level ofvirus replication, methods have been developed to conferregulated-replication or conditional-replication competency toadenoviral vectors based upon complementing, in trans, the essentialearly genes that are missing in the replication-defective vectors. Inthis way, E1-deleted and E4-deleted vectors have been transcomplementedby conjugating them to E1 or E4 expression plasmids. This method enablesthe vectors to replicate, thereby augmenting their transduction ability.Methods have also been explored that allow the continuous replication ofthe vector, such as using the E1a-like activity provided by interleukin6 to enable replication of E1a-deleted vectors.

The present invention further describes methods to enhance thespecificity of the replication of these replicative adenoviral vectors.The current methods of mutating E1, or regulation of E1 withtumor-specific promoters, are both very rational approaches, but mayprove not selective enough for several reasons. In the case of E1deletions, the main limitation lies in incomplete knowledge of the roleof these proteins in the viral replicative cycle and in controlling thecell cycle. For example, adenovirus may use a p53-dependent mechanism torelease the progeny from the infected cell. This would predicate apositive role for p53 in virus production and would reduce the yields ofvirus in p53-deficient cells. On the other hand, other viral proteinsbesides E1-55K may block p53 function, such as E4, and this would allowthe 55K⁻ to replicate in p53+ cells. In any case the specificity of a55K⁻ for p53-defective cells is controversial. Regarding to strategiesbased on regulation of E1 it is a concern that promoters can losecertain degree of specificity when inserted into the viral genome. Thepresence of E1-like activity in uninfected cells could also pose aproblem for the specificity achieved with both vectors. In this regard,some replication of E1 vectors has been observed in many different celllines.

Therefore, it is desirable to improve the replication selectivity ofreplicative adenoviral vectors for tumors by achieving tumor-selectiveregulation of key early genes other than E1, such as E2 or E4. Anadenovirus-polylysine-DNA transcomplementation system has been developedas a means to evaluate replication. This replication-enabling system isused to analyze the efficacy and specificity of tumor-specificreplication mechanisms based on the regulation of the E4 or E2 genes. Inthe transcomplementation system, plasmids encoding E2 or E4 under thecontrol of different tumor-specific promoters are used to screen formechanisms that confer selective replication. Ultimately, selectivereplication will involve the incorporation of the regulated E4 or E2into the viral genome to achieve continuous replication. Accordingly,after the tumor-selective replication has been demonstrated, theseregulatory mechanisms are incorporated into a single viral vector.Optimally, these regulatory mechanisms are combined with the fibermodification described herein to enhance infectivity.

Initial tumor models are based on cell lines with differentialexpression of the PSA protein: LNCaP and DU145. Tumors derived from lungadenocarcinoma cell lines and ovarian cell lines are used to evaluateviruses with promoters such as Carcinoembryonic antigen (CEA) orsecretory leukoprotease inhibitor (SLPI). Therapeutic effects are onlyobserved in tumors derived from the cell lines that allow the expressionof the tumor-specific controlled E4 or E2, that is, replication of thevirus. In these permissive cell lines, higher therapeutic advantage isobserved for the RGD-modified virus relative to the unmodified virus.

The present invention is directed towards an infectivity-enhancedconditionally replicative adenovirus. This adenovirus possesses enhancedinfectivity towards a specific cell type, which is accomplished by amodification or replacement of the fiber of a wildtype adenovirus andresults in enhanced infectivity relative to the wildtype adenovirus. Theadenovirus also has at least one conditionally regulated early gene,such that replication of the adenovirus is limited to the specific celltype. Preferably, the cell is a tumor cell.

Preferably, the modification or replacement of the fiber results inCAR-independent gene transfer. Generally, the modification isaccomplished by introducing a fiber knob domain from a different subtypeof adenovirus. The fiber can also be modified by introducing a ligandinto the HI loop of the fiber knob, or replacing the fiber with asubstitute protein which presents a targeting ligand. Representativeligands include physiological ligands, anti-receptor antibodies andcell-specific peptides. Additionally, the ligand may comprise atripeptide having the sequence Arg-Gly-Asp (RGD), or more specifically,a peptide having the sequence CDCRGDCFC.

Generally, the fiber substitute protein associates with the penton baseof the adenovirus. Structurally, the fiber substitute protein ispreferably a rod-like, trimeric protein. It is desirable for thediameter of the rod-like, trimeric protein to be comparable to thenative fiber protein of wild type adenovirus. It is important that thefiber substitute protein retain trimerism when a sequence encoding atargeting ligand is incorporated into the carboxy-terminus. In apreferred aspect, a representative example of a fiber substitute proteinis T4 bacteriophage fibritin protein. In a preferred embodiment, thefiber substitute protein comprises: a) an amino-terminal portioncomprising an adenoviral fiber tail domain; b) a chimeric fibersubstitute protein; and c) a carboxy-terminal portion comprising atargeting ligand. More generally, the fiber substitute protein can beselected from the group consisting of trimeric structural proteins,trimeric viral proteins and trimeric transcription factors. Otherrepresentative examples of fiber substitute proteins include isoleucinetrimerization motif and neck region peptide from human lung surfactantD. Preferably, the fiber substitute protein has a coiled coil secondarystructure. The secondary structure provides stability because ofmultiple interchain interactions. The fiber substitute protein does nothave to be a natural protein. In fact, a person having ordinary skill inthis art would be able to construct an artificial protein. Preferably,such an artificial fiber substitute protein would have a coiled coilsecondary structure.

The early gene may be conditionally regulated by means consisting of atissue-specific promoter operably linked to an early gene (e.g., E1, E2and/or E4) and a mutation in an early gene (e.g., E1, E2 and/or E4).Representative tissue-specific promoters are derived from genes encodingproteins such as the prostate specific antigen (PSA), Carcinoembryonicantigen (CEA), secretory leukoprotease inhibitor (SLPI),alpha-fetoprotein (AFP), vascular endothelial growth factor, CXCR4 orsurvivin.

Additionally, the adenovirus may carry a therapeutic gene in its genome.In conjunction with the above-mentioned adenoviral vector, a method ofproviding gene therapy to an individual is disclosed herein, comprisingthe steps of: administering to the individual an effective amount of aninfectivity-enhanced conditionally-replicative adenovirus.Representative routes of administration are intravenously,intraperitoneally, systemically, orally and intratumorally. Generally,the individual has cancer and the cell is a tumor cell. When thetherapeutic gene carried by the adenovirus is, for instance, a herpessimplex virus thymidine kinase gene, the present invention furtherprovides for a method of killing tumor cells in an individual,comprising the steps of: pretreating the individual with an effectiveamount of an infectivity-enhanced conditionally-replicative adenovirusexpressing the TK gene; and administering ganciclovir to the individual.Generally, the individual has cancer.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Therefore, if appearing herein, the following terms shall havethe definitions set out below.

A “vector” is a replicon to which another DNA segment may be attached soas to bring about the replication of the attached segment. A “replicon”is any genetic element (e.g., plasmid, chromosome, virus) that functionsas an autonomous unit of DNA replication in vivo; i.e., capable ofreplication under its own control. An “expression control sequence” is aDNA sequence that controls and regulates the transcription andtranslation of another DNA sequence. A coding sequence is “operablylinked” and “under the control” of transcriptional and translationalcontrol sequences in a cell when RNA polymerase transcribes the codingsequence into mRNA, which is then translated into the protein encoded bythe coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell. A “cis-element” is a nucleotide sequence, alsotermed a “consensus sequence” or “motif”, that interacts with otherproteins which can upregulate or downregulate expression of a specificgene locus. A “signal sequence” can also be included with the codingsequence. This sequence encodes a signal peptide, N-terminal to thepolypeptide, that communicates to the host cell and directs thepolypeptide to the appropriate cellular location. Signal sequences canbe found associated with a variety of proteins native to prokaryotes andeukaryotes.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters containShine-Dalgarno sequences in addition to the −10 and −35 consensussequences.

As used herein, the terms “conditionally regulated” and“conditionally-replicative” refer to the expression of a viral gene orthe replication of a virus or a vector, wherein the expression ofreplication is dependent (i.e., conditional) upon the presence orabsence of specific factors in the target cell.

As used herein, the term “early genes” refers to those adenoviral genesexpressed prior to the onset of adenoviral DNA replication.

As used herein, the term “CAR-independent infectivity” refers to theentry of adenovirus into a cell by receptors different from thecoxsackie-adenovirus receptor (CAR).

As used herein, the term “RGD-integrin interaction” refers to binding ofthe arginine-glycine-aspartic acid (RGD) residues in a peptide tointegrin receptor molecules.

As used herein, the term “replication-competent adenoviruses” refers toan adenovirus capable of replication (i.e., an adenovirus that yieldsprogeny).

As used herein, the term “fiber substitute protein” is a protein thatsubstitutes for fiber and provides three essential features: trimerizeslike fiber, lacks adenoviral tropism and has novel tropism.

When used in vivo for therapy, the adenovirus of the present inventionis administered to the patient or an animal in therapeutically effectiveamounts, i.e., amounts that eliminate or reduce the tumor burden. Aperson having ordinary skill in this art would readily be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of this adenovirus of the present invention. Itmay be administered parenterally, e.g. intravenously, but other routesof administration will be used as appropriate. The dose and dosageregimen will depend upon the nature of the cancer (primary ormetastatic) and its population, the characteristics of the particularimmunotoxin, e.g., its therapeutic index, the patient, the patient'shistory and other factors. The amount of adenovirus administered willtypically be in the range of about 10¹⁰ to about 10¹¹ viral particlesper patient. The schedule will be continued to optimize effectivenesswhile balanced against negative effects of treatment. See Remington'sPharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton,Pa.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics8th Ed (1990) Pergamon Press; which are incorporated herein byreference.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Enhanced Tumor Transduction with Adenoviral Vectors Modifiedwith an Antibody Conjugate

As a first approach towards enhancing the infectivity of adenoviralvectors and to demonstrate the tumor transduction advantage of vectorswith altered tropism over unmodified vectors, an anti-fiber antibodyconjugated to fibroblast growth factor (FGF2) was used. The Fab portionof the anti-knob antibody, 1D6.14, which is capable of blocking theinteraction of the fiber with its cognate cellular receptor, waschemically conjugated to FGF2. The resulting Fab-FGF2 conjugate wascomplexed with adenoviral vectors expressing luciferase orβ-galactosidase reporter genes to compare the transduction efficiency ofthe modified and unmodified vectors. Vector modification increased thelevel of gene expression more than 9-fold, as measured by luciferaseactivity (FIG. 1A), largely due to transduction of a greater percentageof target cells as seen by β-galactosidase staining (FIG. 1B). Thisexperiment clearly demonstrates that a retargeted adenoviral vector canovercome the inefficacious transduction observed in certain cell linestransduced poorly by adenoviral vectors.

To compare the therapeutic effect of an PGF2-modified vector to anunmodified vector in established tumors, the conjugate was then mixedwith an adenovirus expressing HSV-TK (AdCMVHSV-TK). Treatment of SKOV3ovarian carcinomas established in nude mice with the modified vectorfollowed by administration of the prodrug, ganciclovir, resulted in asignificant prolongation of survival when compared with the unmodifiedvector plus ganciclovir (FIG. 2). Thus, retargeting can increase the invivo therapeutic effect of adenoviral vectors against tumors. It isclear that the infectivity of tumors by unmodified adenovirus is notoptimal and modification of the capsid to alter the tropism of the virusis a direct approach to increase this infectivity.

Example 2 Genetic Modification of the Hi Loop of the Fiber ProvidesEnhanced Infectivity to Adenoviral Vectors

The Fab-ligand conjugation method described in Example 1 only modifiesthe tropism of the vector prepared for inoculation. In the context of areplicative vector, it is advantageous to modify the tropism of thevector that replicates in the tumor as well. With this rationale, agenetic modification of the fiber is necessary for replicative vectorsbecause it is carried over to the progeny. As a simple and potentstrategy for retargeting, the sequence of the fiber was geneticallymodified. Based on the three-dimensional model of the fiber knob,targeting ligands were inserted into the HI loop of the fiber (FIG. 3).This loop is flexible, exposed on the outside of the knob, is notinvolved in fiber trimerization and its variable length in different Adserotypes suggests that insertions or substitutions would not affect thefiber stability.

As a ligand to introduce into the HI loop of the fiber knob, thesequence coding for an RGD peptide, CDCRGDCFC (SEQ ID NO:1), was chosen.This RGD sequence is known to target tumors by binding with highaffinity to several types of integrins. It was hypothesized that anadenoviral vector able to bind via fiber-RGD/integrin interaction wouldnot depend upon the presence of the CAR receptor in tumors to beeffective, and would therefore target tumors more efficiently than theunmodified vector counterpart.

The DNA sequence encoding the peptide was cloned into the EcoRV site ofthe knob domain in a plasmid containing the fiber sequence. The wildtype fiber of an E1,E3-deleted adenoviral vector expressing theluciferase gene, AdCMVLuc, was replaced with the RGD-modified fiber byhomologous recombination in bacteria. After homologous recombination,the genome of the new adenoviral vector was released from the plasmidbackbone by digestion with PacI. To use the firefly luciferase gene, theinternal PacI site of this gene was eliminated by introducing a silentmutation. The plasmid obtained as a result of these DNA recombinationswas then utilized for transfection of 293 cells to rescue Ad5IucRGD. Thepresence of RGD in the virus was confirmed by PCR as well as by cyclesequencing of viral DNA isolated from CsC1-purified virions ofAd5lucRGD.

To demonstrate that the genetic modification of the fiber was able toconfer CAR-independent infectivity to the modified vector, theunmodified AdCMVLuc and the modified Ad5lucRGD vectors were used totransduce 293, HUVEC, and RD cell lines, which express high, moderate,and low levels of CAR respectively. The CAR-independent infection wasfurther analyzed using competitive inhibition by recombinant Ad5 fiberknob protein known to efficiently block virus binding to CAR receptor.Luciferase expression in 293 cells mediated by the unmodified virus,AdCMVLuc, was efficiently blocked by recombinant knob protein. Dependingon the multiplicity of infection (MOI) used, knob protein blocked 85% to93% of luciferase activity in AdCMVLuc-transduced cells. In contrast,the same concentration of knob was able to block only 40% to 60% ofAd5lucRGD-mediated gene expression in 293 cells, indicating that inaddition to the fiber-CAR interaction utilized by the wild type Ad5,Ad5lucRGD is capable of using an alternative, CAR-independent, cellentry pathway. Of note, the contribution of that alternative mechanismof cell binding was quite significant, providing 40% to 60% of overallgene transfer to 293 cells. Luciferase expression in HUVEC cellstransduced with Ad5lucRGD was about 30-fold higher than with AdCMVLuc.The effect of Ad5 fiber knob on AdCMVluc-mediated transduction was lessdramatic than in 293 cells, consistent with a relative lack of CAR inthe HUVEC. Most importantly, recombinant knob protein did not inhibitthe levels of luciferase expression directed by Ad5lucRGD. Theluciferase activity detected in RD cells transduced with AdCMVluc wasextremely low: at an MOI of one pfu/cell, it was almost equal to thebackground level of mock-infected cells. In contrast, the level oftransgene expression achieved with Ad5lucRGD was 16- to 47-fold higherthan with AdCMVLuc, and expression was not inhibited by the fiber knob.

These experiments clearly showed that incorporation of the RGD peptideinto the fiber of Ad5lucRGD resulted in dramatic changes invirus-to-cell interaction by providing an alternative CAR-independentcell attachment pathway. Of note, the insertion of the RGD sequence inthe HI loop did not abrogate the CAR-mediated entry pathway, so themodified vector has a two independent mechanism to bind to the cells. Asthe present invention shows, this contributes to the enhancedinfectivity of the modified vector in all cell lines and tumors tested.

Example 3 Enhanced Tumor Transduction Via RDG-Fiber Modification

To determine if the RGD sequence incorporated into the HI loop of thefiber could increase the infectivity of tumors, the ability of themodified vector to deliver genes to cultured human ovarian cancer cellswas examined. Characterization of two cell lines, SKOV3.ipl and OV-4, byflow cytometry showed that they both express moderate-to-high levels ofανβ3 and ανβ35 integrins. SKOV3.ipl also expresses a high level of CAR,whereas OV-4 only modestly expresses CAR.

The incorporation of recombinant RGD-containing fiber protein in theAd5lucRGD vector dramatically improved the ability of the virus toefficiently transduce these cells (FIG. 4A). At different MOIs tested,Ad5lucRGD-transduced cultures of SKOV3.ipl cells showed 30-fold to60-fold increase in luciferase activity compared to cells transducedwith control virus. Interestingly, while the purified fiber knob blockedover 90% of AdCMVLuc-mediated gene transfer, it could only block 20% ofluciferase activity in Ad5lucRGD-treated cells, indicating a significantCAR-independent entry mechanisms for Ad5lucRGD. In OV-4 cells, thetransduction efficiency achieved with the RGD-modified vector was 300-to 600-fold higher than the unmodified one (FIG. 4B). Again, when thefiber knob was used as an inhibitor of CAR-mediated cell entry, it didnot have any significant effect on Ad5lucRGD-mediated gene delivery,strongly suggesting that this virus primarily utilizes RGD-integrininteraction to bind to target cells.

The utility of the Ad5lucRGD vector was next evaluated in the context ofprimary tumor cells. In this regard, recent human clinical trials havepointed out the disparity between the efficacy of adenoviral vectors invarious model systems and in the clinical context, where rather lowtransduction efficiencies have been noted. As integrins have been shownto be frequently overexpressed by various epithelial tumors, vectortargeting to these cell surface receptors provides a means to achieveCAR-independent gene transfer.

Ovarian cancer cells obtained from two patients were treated with eitherAd5lucRGD or AdCMVLuc in the presence or absence of blocking knobprotein. Luciferase expression in cells treated with AdCMVLuc wasextremely low (FIG. 5), thereby indicating inability of adenoviralvectors containing unmodified fibers to efficiently infect ovariancancer cells. Strong inhibition by the fiber knob on AdCMVLuc-mediatedluciferase expression suggests that the fiber-CAR interaction is theonly pathway this virus can use to infect this type of cell. Incontrast, Ad5lucRGD directed levels of transgene expression two- tothree-orders of magnitude higher than those detected inAdCMVLuc-transduced cells. The knob protein blocked 20% of the genetransfer at an MOI of 1 pfu/cell, and no effect was observed at an MOIof 10 pfu/cell.

The observations of enhanced infectivity have been extended to othertumor cell types besides ovarian carcinoma. In six human non-small celllung adenocarcinoma cell lines, one 25 human mesothelioma cell line, andone rat mesothelioma cell line, the luciferase expression level achievedwith the RGD-modified vectors was always higher than the level achievedwith the non-modified vector at a variety of different MOIs.

The increase in transduction was also observed in A549 lungadenocarcinoma cells and LNCaP prostate carcinoma cells (FIG. 6). Inboth cell lines the RGD modified vector showed an infectivity advantageover the non-modified counterpart. The major difference was observed inA549 cells, showing a 100-fold increase in infection, whereas LNCaPcells showed 10-fold increase. In LNCaP, the major differences wereobserved at lower multiplicities of infection, likely indicating thatthe integrin-mediated pathway was saturated.

The increased efficacy of infection of the RGD-modified vector was alsomeasured in time course experiments in which the incubation time of thevirus with the cells was limited. The transduction efficiency was alwaysbetter with the modified vector and the differences were more marked atshorter times of infection, i.e. the RGD-modified vector produced a1000-fold greater luciferase expression when only 7 minutes ofadsorption were allowed. At longer adsorption times, the differencesbetween the modified and non-modified vectors were reduced to 10-fold.This difference could have important implications in adenoviral-mediatedgene therapy because the time of exposure of the vector to the tumortarget cells is expected to be limited by the intratumoral highpressure.

Overall, this data points out the importance of providing an alternativeentry pathway to adenoviral vectors for the infectivity of tumors. Inall cell lines and tumor types analyzed, a vector that can use thenatural entry pathway via primary binding to CAR and an additional entrypathway via binding to integrins transduces more efficiently than avector that only can use the natural CAR receptor.

Example 4 Replication-Competent, E1-Transcomplementation Vectors

Most replication-defective adenovirus vectors in preclinical andclinical use have deleted E1A and E1B genes. These deletions render thevector unable to replicate, or replication-incompetent, and thesevectors can replicate only when E1 proteins are supplied in trans. Thesereplication-incompetent vectors transduce the cells that they infect butthey do not produce any progeny.

A conditional replication enablement system for adenovirus has beendeveloped in which the E1 genes are supplied in trans to cells infectedwith E1-deleted vectors (FIG. 7). The replication-enabling system hasbeen developed primarily as a means of amplifying transduction in tumornodules. In order to achieve a more extensive amplification of thevector and lysis of tumor cells, the secondarily produced vector shouldpropagate continuously in tumor cells. Replication-enabling has beenachieved by linking plasmids encoding the E1 proteins to the exterior ofthe capsid or separately introducing the plasmid using cationic lipids.These experiments provided evidence that replication-enabling systemscould achieve amplification of the in vivo therapeutic response of anadenoviral vector carrying HSV-TK. E4-deleted adenoviruses have alsobeen transcomplemented with a plasmid containing the E4 open readingframe 6 gene or the complete E4 region. E4 transcomplementation isimportant in the context of reducing immunogenicity and increasinglong-term gene transfer.

In order to further enhance the utility of the replication-enablingsystem, it is a goal of the present invention to reduce thepossibilities of recombination between the E1-deleted vector and thetranscomplementing plasmid. This recombination would generatereplication competent adenoviruses (RCA). Therefore, an E1 expressingplasmid has been constructed, pE1FR, in which E1a and E1b sequences arein tandem but oriented in opposite 5′ to 3′ direction. Cellsco-transduced with this plasmid and an E1-defective adenoviral vectorusing cationic liposomes resulted in replication-defective adenovirusproduction levels comparable to that achieved by co-transduction of thevirus and pE1 (FIG. 8). Comparable results were obtained with HeLa, A549and SKOV3-ipl cell lines.

This demonstrates that pE1FR can transcomplement E1-deleted vectors andconvert the infected cells into vector-producing cells. To demonstratethat this vector could also enhance the tumor transduction achieved withan E1-deleted vector in vivo, tumors were injected with E1-defectivevirus mixed with pE1FR, or a plasmid control. Assessment of theluciferase content showed that 6 out of 8 tumors had increasedluciferase activity in the pE1FR group relative to the controls (FIG.8).

This data indicates that E1-expression vectors, such as pE1FR, representa feasible way to increase the in vivo transduction efficiency ofE1-deleted vectors in tumors. The amplification of the transductionefficiency achieved with a system such as the replication enablingsystem is limited. however, by the inability of the vector progeny tokeep replicating. The replication-enabling function needs to be carriedover in the vectors produced by the tumor cells to allow repeated cyclesof replication.

Example 5 Replication Competent Vectors Dependent Upon IL-6

As shown in the data above, the replication-enabling system has beendeveloped primarily as a means of amplifying transduction in tumornodules. Methods have also been explored to achieve a more extensiveamplification of the vector and subsequent lysis of tumor cells. Tofulfill this goal, the secondarily produced vector should propagatecontinuously in tumor cells and incorporate a regulatory mechanism thatconfines this propagation to the tumor. E1a 12s and 13s adenoviralproteins are necessary to induce the expression of other viral genes,and therefore, an E1a-deleted vector is impaired in its replication. Ithas been reported that interleukin 6 can induce transcription factorsthat are able to substitute for the E1a activity of adenovirus.

To explore whether an E1a-deleted vector such as Ad5dl312 couldreplicate in the presence of IL-6 in different cancer cell lines, cellswere infected with dl312 in the presence of IL-6 and the progeny wereexamined (FIG. 10). In all cell lines, infectious virions were producedto a certain extent in the presence and absence of IL-6, although inlower amounts than the wild type adenovirus. The effects of IL-6 indl312 production were markedly seen in two cell lines: HepG2 and EJ. InHepG2 cells, IL-6 resulted in a 1.5 log increase of viral production.

These experiments demonstrate that the IL-6-inducible E1a-like activitycan complement the E1a deletion during infection of HepG2 and EJ cells.To overcome the requirement of exogenous IL-6, carcinomas, e.g.,cervical, chorio, and ovarian, that have an IL-6 autocrine loop wereinfected with the E1A-deleted virus, dl312. OVCAR-3 and SW626 cells havea functional IL-6 autocrine loop. Upon infection of OVCAR-3 cells withAd5dl312, or wild type or E4-deleted control viruses, Ad5dl312 wasproduced to levels similar to levels produced by the wild type control,even in the absence of IL-6 (FIG. 11). This IL-6 independent replicationof E1a-deleted virus was also demonstrated in SW626 cells and primarycultures of ovarian tumors (FIG. 11).

These results indicate that cells with an autocrine loop of IL-6 canselectively support the replication of Ad5dl312 without the addition ofexogenous IL-6, and that these cells are lysed by the E1a-deleted virus.The effects of the E1a-deleted virus in normal cells were examined. Totest the ability of this virus to propagate in normal cells adjacent toovarian tumors, human mesothelial cells isolated from peritoneal liningtissue were infected. Contrary to the wildtype virus control, Ad5dl312did not replicate in these cells even in the presence of IL-6.

Overall, this data indicates that E1a-deleted adenovirus can becomplemented by the IL-6-induced E1a-like activity found in severaltumors. E1a-deleted vectors are, however, limited by the fact that E1aintrinsic activity has been noted in normal cells. IL-6 production, inthe other hand, could result from the injection of the vector in animmunocompetent host and this natural inflammatory response would resultin nonspecific complementation. Clearly, new mechanisms oftumor-specificity need to be incorporated to control the replication ofadenoviral vectors.

The clinical benefits of cancer gene therapy achieved withnon-replicative adenoviral vectors have been hampered by the significantnumber of cells in a tumor which have been left unaffected by the director indirect effects of the transgenes. Conditional replicativeadenoviruses may represent a significant improvement to solve thisproblem, but efficient infectivity and tumor-selective replication needto be achieved to realize their full potential.

The importance of the modification of the adenoviral capsid to increasethe binding of the vector to the tumor cells has been demonstratedherein. An integrin-binding RGD motif inserted in the HI loop of theadenoviral fiber confers an additional binding pathway besides thenatural coxsackie-adenovirus receptor, and this dramatically increasesthe infectivity of the vector. The data herein also indicates thattransduction efficiency can also be enhanced if the vector is able toreplicate in the tumor. A transcomplementation system has been developedas a means to evaluate the effects of replication on the transductionefficiency. This replication-enabling system also provides theopportunity to analyze the efficacy and specificity of differenttumor-specific replication mechanisms before incorporating thesemechanisms into a single viral vector in a cis-complementation strategythat will allow continuous replication. In this regard, continuoustumor-selective replication has been shown using E1a-deletion mutantsthat propagate in tumors due to an E1a-like activity.

Example 6 Incorporation of RGD-Fiber into Currently Defined ConditionalReplicative Mutant Viruses

As an initial approach towards comparing the therapeutic potential of anRGD-modified versus an unmodified replicative adenovirus, conditionalreplicative mutants that have been previously described were chosen.Deletion of the E1b-55K protein was designed to confer selectivereplication to adenoviruses in cells lacking functional p53. In asimilar way, deletion of the Rb-binding sites of E1a has been proposedto achieve selective replication in cells lacking Rb. These deletionmutants are used as established models of selectivereplication-competent viruses.

The initial plasmid to construct these deletions is pXC1, which containsadenoviral sequences from basepair 22 to 5790 (Microbix, Hamilton,Canada). For the E1b55K deletion, the region from Sau3AI (Ad5#2426) toBglII (Ad5#3328) is removed by ligation of the 1 kb Xba1-Sau3AI DNAfragment with the 7.9 kb XbaI-BglII DNA fragment to yield plasmidpXC-55K-. For an E1a deletion construct that abrogates binding to Rb, aderivative of pXC1 (pXC1 D24) is obtained with E1a deleted in residues122 to 129 (Dr. Juan Fueyo, MDACC). This deletion affects the residuesof the conserved region 1 of E1a necessary to bind Rb. These E1b and E1adeletions are incorporated into the viral genome by homologousrecombination with plasmid pVK503, containing either an unmodified fiberor an RGD modified fiber. From the plasmids obtained by homologousrecombination, the unmodified 55k- and D24 mutants are generated byreleasing the viral genome with PacI and transfecting into 293 cells.Viruses are amplified and purified by double CsC1 gradient, and titeredin 293 cells for in vitro and in vivo experiments. The presence ofmutated E1, altered fiber, and contaminating wild type E1, is analyzedby PCR as well as by sequencing of viral DNA isolated from CsC1-purifiedvirions.

The 24-bp deletion in the E1A gene and the RGD encoding sequence in thefiber were verified by PCR (FIG. 12). The presence of the RGD motif inthe modified fiber was confirmed by PCR employing fiber primers FiberUp(5′-CAAACGCTGTTGGATTTATG-3′) (SEQ ID NO:2) and FiberDown(5′-GTGTAAGAGGATGTGGCAAAT-3′) (SEQ ID NO:3). The 24-bp deletion wasanalyzed by PCR with primers E1a-1 (5′ATTACCGAAGAAATGGCCGC-3′) (SEQ IDNO:4) and E1a-2 (5′CCATTTAACACGCCATGCA-3′) (SEQ ID NO:5) followed byBstXI digestion. Of note, no adenoviruses having wild-type E1 orwild-type fiber appeared throughout the propagation of Ad5-D24RGD, afinding that confirms the lack of endogenous adenoviral sequences inA549 cells.

Example 7 Evaluation of Infectivity of RGD-Modified ConditionalReplicative Viruses

Procedures described above are used to demonstrate that the RGD-modified55K- and D24 virions bind to integrins. ELISAs are performed withimmobilized virions incubated with purified ανβ3 integrins and anti-αsubunit monoclonal antibody, VNR139. The modified replicative virusesare examined to determine if they are able to bind cells via aCAR-independent pathway. 293, HUVEC, and RD cells are used, as enhancedRGD-mediated transduction of these cell lines has already beendemonstrated. For binding analysis, virions are labeled with ¹²⁵I andincubated with cells. Recombinant knob protein is used as an inhibitorto measure CAR-independent binding. Infectivity of modified andunmodified 55K and D24 mutants in ovarian, lung and other tumor celllines, as well as in primary tumors, are compared. These experimentsindicate that the RGD-modified viruses infect tumor cells moreefficiently than the non-modified vectors.

Example 8 Evaluation of Oncolytic Potential of RGD-Modified ConditionalReplicative Viruses

This example demonstrates that the genetic introduction of an RGDsequence in the fiber of a CRAd allows CAR-independent infection thatleads to the enhancement of viral propagation and oncolytic effect invitro and in vivo.

Cell Lines

A549 human lung adenocarcinoma and LNCaP human prostate cancer celllines were obtained from the American Type Culture Collection (Manassas,Va.). The cells were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 5% heat-inactivated fetal bovine serum (PBS),100 I.U./ml penicillin, and 100 mg/ml streptomycin.

Virus DNA Replication

A549 cells cultured at 90% confluence in 6-well plates were infectedwith Ad5-D24 or Ad5-D24RGD at a dose of 0.01 viral particles/cell. After2 h, the cells were washed and maintained in DMEM-5% FBS with 1 mCi/mlbromodeoxyuridine (BrdU) (Amersham Pharmacia Biotech Inc., Piscataway,N.J.). Attached and detached cells were harvested at 2, 4, 6, and 8 daysafter infection, and encapsidated viral DNA was purified by thespermine-HCl method (Hardy et al., 1997). One third of the totalpurified viral DNA (corresponding to 6×10⁵ cells) was digested withHindIII and resolved in 1% agarose gel. The fragments were transferredto a nylon membrane (Amersham Pharmacia Biotech), fixed, blocked inblocking buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% dry milk, 2%Tween 20), and incubated with mouse anti-BrdU IgG (DAKO, Carpinteria,Calif.) at 4° C. overnight. The membrane was washed next day, incubatedwith peroxidase-labeled anti-mouse antibody (Amersham), and processed byWestern blotting analysis with the ECL system (Amersham). The membranewas exposed to Kodak Biomax ML film for 3 seconds at room temperatureand developed in an automated processor.

Adenovirus Yield Assay

A549 cells cultured at 90% confluence in 6-well plates were infectedwith 0.01 particles/cell of Ad5lucRGD, Ad5-D24, or Ad5-D24RGD for 2 h.The cells were then washed thoroughly with PBS to remove allnon-adsorbed viruses, and maintained in DMEM 5% FBS. After 8 days, cellsand media were harvested, freeze-thawed 3 times, centrifuged, and thetiter was determined by plaque assay with A549 cells as targets.

Oncolysis Assay

A549 and LNCaP cells cultured in triplicate in 6-well plates wereinfected with one of the three types of adenovirus at doses of 0.001 or0.01 viral particles/cell when 90% confluence was reached. Eight or tendays after infection, the cell monolayers were washed with PBS, fixedwith 10% fresh buffered formaldehyde for 10 min, and stained withcrystal violet solution (1% crystal violet [w/v], 70% ethanol). After 1h staining, the plates were rinsed with tap water and dried.

In Vitro Cytotoxicity Assay (XTT)

A549 and LNCaP cells were seeded and infected in parallel with the onesused for the oncolysis assay described above. Eight or ten days afterinfection, the media was carefully removed, and fresh media containing200 μg/ml of2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide(XTT) (Sigma, St. Louis, Mo.) was added. Cells were then incubated for 3h at 37° C. The content of each well was transferred to a microwellplate, and the light absorbance was read at 450 nm in a microplatereader (Molecula•Devices Corp., Sunnyvale, Calif.). The number of livingcells was calculated from non infected cells cultured and treated withXTT in the same way as were the experimental groups.

Subcutaneous Tumor Xenograft Model in Nude Mice

Female athymic nu/nu mice (Frederick Cancer Research, MD) 8-10 weeks oldwere used to grow A549 s.c. nodules. Eight million cells werexenografted under the skin of each flank in anesthetized mice. When thenodules reached 60-100 mm³ a single dose of 10⁹ viral particles(high-dose experiment, n=5) or 10⁷ viral particles (low-dose experiment,n=4) of Ad5lucRGD, Ad5-D24, Ad5-D24RGD, Ad5-wt or PBS was administeredintratumorally (i.t.). Tumor size was monitored twice a week, andfractional volume was calculated from the formula:(length×width×depth)×½. The mice were euthanized 35 days after thetreatment because of the size of the tumors in the control group.Statistical differences among groups were assessed with student's ttests.

Adenovirus Hexon Immunodetection

The presence of adenovirus hexon in the treated tumor xenografts wasassessed by immunofluorescence at the end of the experiment. Frozen A549nodule specimens were sections, fixed in 3% formaldehyde, and blockedwith normal donkey serum for 30 min at room temperature. Then goatanti-hexon antibody (Chemicon Inc., Temecula, Calif.) was applied for 2h at room temperature, followed by PBS rinse and incubation with AlexaFluor 488-labeled donkey anti-goat antibody (Molecular Probes, Eugene,Oreg.) for 30 min at room temperature. The slides were then rinsed andcounterstained with Hoechst 33342 (Molecular Probe) for 10 min, andanalyzed under a fluorescent microscope (Leitz Orthoplan).

After structural confirmation, the replication capacity of Ad5-D24RGDand Ad5D24 were compared in A549 cells. Cell monolayers were infectedwith low dose of each virus (0.01 viral particles/cell), and weremaintained in media with BrdU throughout the 8-day incubation period.The encapsidated viral DNA was purified on days 2, 4, 6, and 8postinfection. Viral DNA corresponding to 6×10⁵ cells was analyzed bySouthwestern blot using anti-BrdU antibody. As indicated by the BrdUincorporated into replicating viral DNA, Ad5-D24RGD propagation was moreefficient than that of Ad5-D24 (FIG. 13). The Ad5D24RGD DNA can bedetected not only sooner (day 6) compared to Ad5-D24 DNA (day 8), but ingreater amounts. Thus, the infectivity advantage conferred by RGDincorporation into the fiber knob increased adenovirus propagation intarget cells. As this tropism modification would not be anticipated toalter fundamental aspects of the viral replication cycle, this effectwas likely achieved exclusively on the basis of the infectivityenhancement allowed by routing the virus through CAR-independentpathways.

Based on the previous experiment, the actual amount of infectious virusproduced by Ad5lucRGD, Ad5-D24, or Ad5-D24RGD in A549 cells at 8 daysafter infection were quantified by plaque assay. Ad5-D24RGD produced aviral yield of 3.75×10⁹ pfu/ml, which was 43 times higher than that ofits unmodified Ad5-D24 counterpart (8.75×10⁷ pfu/ml). No virus wasobtained from the nonreplicative control Ad5lucRGD infected cells. Theseresults are consistent with the fact that modifying the fiber knob withan RGD motif led to enhancement of viral infectivity and an increase inthe production of infectious adenovirus.

To demonstrate the increased lytic potency of Ad5-D24RGD, A549 and LNCaPcells were infected with small amounts of each virus to allow multiplecycles of viral replication over the ensuing 8 days, then stained theattached cells with crystal violet and counted viable cells by XTTassay. In both cell lines, the fewest viable cells were detected in theAd5-D24RGD-infected group (FIG. 14). The cell lysis capacity ofAd5-D24RGD is 7 times higher in A549, and 3.5 times higher in LNCaPcompared to Ad5-D24. These results demonstrate that the fiber knobmodification enhanced adenoviral lytic potency over that of the Ad5-D24virus.

A goal of this study was to support the oncolytic superiority ofinfectivity enhanced conditionally replicative adenovirus (CRAd) overthat of unmodified adenoviruses in vivo. Since low doses of virus allowseveral cycles of replication along with destruction of tumor cells,even a single dose would produce an exponential rise in the number ofkilled cells, which would extend to the entire tumor. In order todemonstrate this hypothesis, A549 xenografts in nude mice were treatedwith a single i.t. injection (10⁹ viral particles) of one of the threeviruses or with PBS. At 32 days after injection, both CRAds demonstratedto have an oncolytic effect in the tumors opposite to those treated withnonreplicative virus or with PBS (Ad5-D24, p<0.05; Ad5-D24RGD, p<0.01compared to PBS group) (FIG. 15A). Given these results, anotherexperiment was performed in which a 100-fold lower dose (10⁷ viralparticles) of the viruses were administered. This low-dose treatmentdemonstrated that the oncolytic effect of Ad5-D24RGD was superior tothat of Ad5-D24 (p<0.05). These differences observed between high-doseand low-dose experiments suggest that a threshold dose over 10⁷ viralparticles of Ad5-D24 is required to obtain an oncolytic effect in tumornodules (FIG. 15B).

To confirm that the CRAds were present in the tumor tissue,immunofluorescence assays were used to detect the virus hexon in tumorsamples collected after the low-dose experiment (35 days postinjection).Ad5-D24RGD was present in the tumor nodules, as was Ad5-D24 to a lesserextent. PBS and Ad5lucRGD treated nodules showed no hexon signal (FIG.15C). These results corroborate that the partial reduction of tumor masswas due to virus replication and that the RGD modification of the fiberknob conferred infectivity and oncolysis advantage to a CRAd in vivo.

Enhanced oncolytic potential was also demonstrated in a systemiccontext. A total dose of 10⁹ viral particles divided into twoconsecutive doses of 5×10⁸/day of either Ad5lucRGD, Ad5-D24, Ad5-D24RGD,Ad5-wt, or PBS were injected in the tail vein of nude mice bearing s.c.A549 xenografts. FIG. 16 shows that modification of the fiber to broadenthe tropism of a replicative adenovirus improves the oncolytic potentialin a systemic delivery context.

Conditionally replicative adenoviruses (CRAds) are novel and promisingagents for cancer therapy. However, their efficacy is predicated uponefficient tumor infection, specific replication, and lateral spread. Thedeficiency of coxsackie-adenovirus receptor (CAR) in a variety of tumortargets is a limitation to adenovirus infection. In a previous report,it was demonstrated that the insertion of an RGD motif into the HI loopof the fiber knob of non-replicative adenoviruses enhances tumorinfection, indicating that CAR-independent entry represent a viable wayto circumvent CAR deficiency in some tumor types.

In this example, it was demonstrated that the genetic introduction of anRGD sequence in the fiber of a CRAd allows CAR-independent infectionthat leads to the enhancement of viral propagation and oncolytic effectin vitro and in vivo. The increased initial virus entry into the cellsrendered by the RGD-modification results in sooner detection andaugmented yields of encapsidated DNA of Ad5-D24RGD compared to theunmodified Ad5-D24 (FIG. 13). As this tropism modification is notanticipated to alter fundamental aspects of the viral replication cycle,this effect was likely due to the infectivity enhancement allowed bydelivering the virus through CAR-independent pathways. Subsequently,studies of the oncolytic potency of CRAds in two cell lines concludethat Ad5-D24RGD potency is higher than that of the unmodified virus.Although the XTT assay was not sensitive enough to demonstrate the lyticeffect of Ad5-D24 compared to the non-replicative Ad5lucRGD, the crystalviolet showed early comet-like cytopathic areas in Ad5-D24-treated A549and LNCaP cells, indicating the presence of an incipient lytic effect,whereas Ad5lucRGD treated cells were intact (FIG. 14). The less notabledifference between Ad5-D24RGD and Ad5-D24 seen in LNCaP cells isexplained by the absence of the ανβ3 integrins, compensated by thepresence of other types of RGD-binding integrins (a₃b₁ and a₅b₁) thatwere rapidly saturated (FIG. 14).

Another object of the present invention was to demonstrate the superioroncolytic effect of Ad5-D24RGD in an in vivo model. To this end, A549cells xenografted in nude mice were treated with single, high dose (10⁹viral particles) i.t. injections of Ad5lucRGD, Ad5-D24, Ad5-D24RGD, orPBS, and the results showed that both CRAds (modified and unmodified)yielded similar oncolysis (FIG. 15A). However, when a 100-fold lowerdose (10⁷ viral particles) was administered, it became clear that theoncolytic effect of Ad5D24RGD was higher than that of Ad5-D24 (p<0.05)(FIG. 15B). Furthermore, the observed oncolytic effects were correlatedwith the presence of virus progeny in the tumor samples byimmunofluorescent detection of adenoviral hexon. Hexon was not detectedin PBS (not shown) and Ad5lucRGD treated nodules (FIG. 15C, a), whereasit was detected throughout the tumors treated with CRAds. The comparisonbetween the two CRAds showed that fluorescence in Ad5-D24RGD treatedtumors was stronger than the one observed in Ad5D24 treated tumors (FIG.15C, b and c). The lack of fluorescent staining in tumors treated withthe non-replicative control Ad5lucRGD indicates that the detected hexonbelongs to the viral progeny of Ad5-D24 and Ad5-D24RGD, and not to theinitial inoculum. As regards to the high divergence of the volumes ofPBS and Ad5lucRGD treated tumors, factors such as highly heterogeneouscell replication rates and hypoxic and necrotic areas are known toaffect individual tumor volume after a critical size is reached. Thesedifferences have been noted before when using oncolytic viruses.Nevertheless, total resolution of the tumors in the s.c. xenograft modelwas seen only in some nodules treated with Ad5-D24RGD, indicating thatadministration volume and schema adjustments, such as the ones suggestedrecently by Heise and co-workers (1999), might be necessary to achievecomplete oncolysis.

As presented here and elsewhere, the efficacy of replication-competentviruses employed as oncolytic agents can be improved at the level ofinfectivity. As other tumor binding peptides are isolated (Shinoura etal., 1999; Koivunen et al., 1999), modifications in addition to the RGDinsertion can be considered as well. Of note, the RGD-modificationdescribed here does not preclude the binding of the fiber to CAR, andthe modified virus can enter the cells through a_(v) integrins and CAR.

One approach to improve specific tumor infection/transduction would bethe combination of CAR-ablation and tumor-specific ligands to redirectthe virus tropism. Recently, the adenovirus fiber amino acids crucialfor CAR-binding abrogation and new tumor-selective peptides have beendefined (Koivunen et al., 1999; Roelvink et al., 1999). This combinationwill generate truly targeted viruses, and the efficiency of theirpropagation will depend on the amount of the targeted receptor in thesame way as the propagation of the unmodified virus depends on CAR. Thisstrategy could be very valuable when the population to be targeted ishomogeneous, such as endothelial cells of tumor vasculature.

Other aspects of adenovirus biology that can be improved are replicationspecificity, tumor cell killing, and evasion from host immune responses.Tumor selectivity has been the major area of research with the design ofCRAds based on deletions of adenoviral early genes and utilization oftumor-specific promoters. With regard to cell killing capacity, thecombination of oncolysis with suicide genes such as cytosine deaminaseand herpes simplex: virus thymidine kinase has demonstrated to besuperior to either treatment alone. In a similar way, the combination ofoncolysis with radiotherapy and chemotherapy has also proved to havebetter efficacy. Immune responses will play an important role in theultimate outcome of oncolytic virotherapy, an ideal scenario would favora response that can destroy tumor cells, and yet allow viral spread. Themanipulation of the immune response against adenovirus towards a Th1type could lead in this direction. The use of immunocompetent animalswill be needed for the study of immune response to adenovirus, and alsoovine and canine adenovirus could be useful for this purpose.

Specifically targeted CRAds have theoretical attributes that could makethem effective via systemic administration: low toxicity due to lack ofadsorption and replication in normal cells and low effective dose due totheir amplification. To ascertain whether these agents have enoughtargeting/amplification potency to be efficacious through systemicadministration, the oncolytic efficiency of enhanced infectivity CRAdsadministered via tail vein in mice would be determined. It seems thatnot only the presence of CAR and a_(v) integrin are important foradenovirus infection, but anatomical and immunological barriers are alsocrucial when considering this route of administration. In particular,vector clearance by liver macrophages is a major obstacle that has to beovercome. This can be attempted with targeting or other strategies thatchange the physico-chemical properties of the virion such as PEGylation.The emerging picture is that of a targeted adenovirus that remains incirculation for a sufficient period to achieve specific recognition ofthe target. In such a scenario, the infectivity enhancement maneuversdescribed herein will clearly improve the therapeutic gain achievablevia CRAds.

Example 9 Targeting Endogenous Receptors with ChimericReplication-Competent Adenovirus Vectors

Squamous cell carcinoma of the head and neck (SCCHN) expressesrelatively low levels of the primary adenovirus type 5 (Ad5) receptor,coxsackie-adenovirus receptor (CAR). This relative deficiency of CAR haspredicated the development of CAR-independent transduction strategies tomake adenovirus-mediated cancer gene therapy more efficient for thisdisease. CAR-independent transduction strategies have been made by anumber of methods including the development of adenovirus vectorscontaining chimeric knob domains that alter the virus target celltropism. Recently it has been suggested that the receptor for adenovirustype 3 (Ad3) is more highly expressed in SCCHN compared to the Ad5receptor, thereby making the Ad3 receptor as excellent alternativetarget for SCCHN. Therefore, it is hypothesized that a chimeric Ad5vector containing Ad3 knob domains would have preferential targeting toSCCHN compared to an Ad5 vector containing only Ad5 knob domain.

SCCHN cells were infected with equal amount of two oncolytic Ad5vectors, Ad5luc3 or Ad5/3Luc3. Ad5Luc3 contains an Ad5 knob domain thatnecessitates CAR-dependent transduction. Alternatively, Ad5/3Luc3contains an Ad3 knob domain that utilizes a CAR-independent pathway. Theapparent disproportion of Ad5 receptors and Ad3 receptors on this tumortype resulted in more efficient infection and replication of Ad5/3Luc3compared to Ad5Luc3. The ability of Ad5/3Luc3 to more efficiently infectand replicate resulted in a dramatic increase in the oncolytic effect ofthis virus. Thus, infectivity-enhancement via knob chimerism alsoimproves the oncolytic potency of the CRAd therapy.

Example 10 Evaluation of Tumor-Selective E2 And E4 Functions

One goal of the present invention is to demonstrate that tumor-selectiveregulation of E4 and E2 can confer tumor-selective replication toadenovirus. It has previously been shown that E4-deleted adenovirusescan be transcomplemented by conjugating an E4 expression plasmid intotheir capsid. In this regard, plasmids such as pCEP-ORF6, that containthe E4 ORF6 under a constitutive promoter, can be used totranscomplement E4 deleted viruses, such as dl1014. In order to achievetumor-selective expression of E4-ORF6, tumor-specific promoters aresubstituted for the CMV promoter. Among several tumor or tissueselective promoters that have been used in restricting expression ofgenes to tumor cells, the promoter of the prostate specific antigen(PSA) is used initially. PSA is expressed in prostate cells and has beenused to direct expression of TK to prostate tumors. This promoter waschosen to control E4 and E2 in the context of replicative adenovirusesbecause it has already been used to control E1 in this context (obtainedfrom Dr. Chris Baigma). The promoter is subcloned in front of the E4ORF6in plasmid pCEPORF6 to obtain a pPSA-ORF6 expression plasmid. Toevaluate the conditional replicative phenotype of a PSA-ORF6-regulatedvirus, this plasmid is conjugated with the E4-deleted virus, dl1014.Conjugates with pCEP-ORF6 or irrelevant plasmids are used as positiveand negative controls, respectively. These Ad5dl1014adenovirus-polylysine-plasmid conjugates are used to infect tumor celllines that express prostate specific antigen, such as LNCaP, and celllines that do not express prostate specific antigen, such as DU145 orPC3. In time course experiments, viral replication is measured at theDNA level by Southern blot. The amount of virus produced from themolecular conjugates is measured by plaque assays in W162 cells. dl1014DNA replication and virus production is observed in all cell lines whenusing pCEP-ORF6, but only in the PSA-expressing cell line, LNCaP, whenusing pPSA-ORF6. These results indicate that the E4 can be used tocontrol the replication of E4-deleted adenoviruses and the PSA promoterrestricts this replication to cells expressing PSA.

As a reference background and for comparison purposes, a PSA-E1 plasmidis constructed as a derivative of the E1 constructs used in thereplication-enabling system, such as pE1FR. An E1-deleted vector and 293cells are used to evaluate the selective replication conditional to theexpression of prostate specific antigen. The differential propagationand the levels of virus production obtained with PSA-E4 and PSA-E1regulation indicates which of these regulatory mechanism renders betterselectivity of replication when used independently.

A similar strategy is followed to achieve selective expression of E2.E2-expression plasmids transcomplement E2-defective viruses using thereplication-enabling system. The function of the three open readingframes of E2 (DNA binding protein, terminal protein, and polymerase) aresubcloned into separate plasmids. These open reading frames of E2 arethen placed under the regulation of the PSA promoter. AppropriateE2-defective mutant viruses, such as Ad5ts125 which contains atemperature-sensitive mutation of E2-DBP, are used to construct thecorresponding adenovirus-polylysine-DNA conjugates. As above, theseconjugates are used to infect LNCaP, DU145 and PC3 cell lines. Viral DNAreplication is measured by Southern blot. Cell lines expressing E2 areused to measure the amount of E2-deleted viruses produced by plaqueassays.

Example 11 Construction of RGD-Fiber Adenoviruses with Tumor-SelectiveE4 or E2 Transcriptional Units

It is a goal of the present invention to combine the fiber modificationwith the replication-regulatory mechanisms. Towards this direction, theE4 and/or E2 construct(s) that demonstrated conditional regulation inthe replication-enabling system replace the endogenous viral E4 and/orE2 transcriptional unit. For this, the region that is to be modified issubcloned into a small plasmid to facilitate its manipulation. Thisregion is then removed from the plasmid and co-transformed intocompetent bacteria with a plasmid containing the complete viral genome.The recombination between the viral sequences flanking the modifiedregion and the homologous sequences in the larger plasmid results in theincorporation of the modified region into the adenoviral genome. Beforethe co-transformation step, it is necessary to cut the large plasmid ina unique site located in the middle of the homology region to avoid thepresence of colonies derived from the large plasmid. As there are noavailable unique sites in the E4 or E2 promoter region, theRecA-assisted cleavage method will be used to restrict in the propersite.

This method involves three steps: first, an oligonucleotide spanning thesite to be cut in the E2 or E4 promoter region is annealed to the largeplasmid in the presence of RecA protein (New England Biolabs, Beverly,Mass.) to form a three-stranded segment. Second, a methylase recognizingthis site is then used to methylate all the sites in the large plasmidexcept the one protected by the oligonucleotide. Finally, theoligonucleotide is removed by heat and the corresponding restrictionendonuclease is used to cut the unique non-methylated site. Commonsite-specific methylases, such as AluI, HaeIII, HhaI, HpaII, etc, andthe corresponding restriction endonucleases are purchased from NewEngland Biolabs. Plasmids containing the wild type fiber and plasmidswith the modified RGD fiber are used. After the homologous recombinationstep, the larger plasmids containing the viral genomes with thesubstituted E4 or E2 regions are cut with PacI release the viral genome.Finally, the viruses are obtained by transfection into E4 or E2complementing cell lines. Viruses are amplified and purified by doubleCsC1 gradient, and titered in these cell lines for in vitro and in vivoexperiments. The presence of the E4 or E2 transcription unit regulatedwith the tumor-specific promoter and of the mutated fiber is analyzed byPCR as well as by sequencing of viral DNA isolated from CsC1-purifiedvirions.

Example 12 Testing of Adenoviruses with Enhanced Infectivity andTumor-Selective Replication

Mice containing human tumors can be used to evaluate the therapeuticpotential of adenoviruses with enhanced infectivity and tumor-selectivereplication. Three types of models can be used: subcutaneous engraftedcell lines (e.g. prostate LNCaP and DUI45), diffuse intraperitonealengraftments (e.g. ovarian SKOV3-ip 1), and liver metastases (e.g.colorectal carcinoma cell line LS174T). Adult (6-8 week old) athymicnu/nu mice can be used in the subcutaneous and metastatic models whereasSCID mice can be used in the intraperitoneal model. Except for theprostate cell lines, female mice are used. Treatments include theRGD-modified, non-modified and vehicle control in a single injection foreach dose. Intratumoral, intraperitoneal or intravenous administrationof the viruses (according to the model used) is performed with unsedatedmice using gentle physical restraint. All mice are euthanized at theconclusion of all experiments by CO₂ vapor sedation followed byPhenobarbital overdose.

Localized Models

Subcutaneous tumor nodules can be established using the LNCaP and DU145cell lines. Cells (10⁷) are mixed 1:1 with Matrigel (CollaborativeBioproducts), loaded into syringes and injected subcutaneously in atotal volume of 200 μl into the front flanks of athymic nude mice (2×10⁶cells per engraftment site). Initially, three pairs of viruses arecompared: PSAE4-RGD versus PSAE4; PSA-E2 versus RGD-PSAE2; and PSA-E1versus RGD-PSAE1. Viruses with double E1/E4 or E1/E2 controlledtranscriptional units can also be analyzed. Tumor nodules are injectedwith the appropriate adenovirus or vehicle control (PBS/10% glycerol)when their volume (length×width 2×½) reaches 0.2 mm³. Injections arewith a Hamilton syringe in a volume of 20 μl ( 1/10 of tumor volume).The amount of virus injected per tumor is adjusted from 10⁴ pfus (plaqueforming units) to 10⁸ pfus by serial dilution. A series of experimentsare done to measure the tumor volume until regression or a maximum of 1cm³. Another series of experiment are performed to measure theintratumoral amount of virus in a time course. This amount is measuredby resecting the tumors and staining sections with anti-hexon antibody(Chemicon) and by extracting the virus from the tumors and measuring theviable virus in a plaque assay. In DU145 tumors, no therapeutic effectis observed with the PSA-controlled viruses. In LNCaP tumors, smallertumors or complete tumor regressions is observed, and more intratumoralvirus in tumors treated with the PSA-controlled replicative viruses isobserved when compared to the non-replicative and vehicle controltreated tumors. Smaller tumors or more frequent complete regressions areobserved, likely due to higher amounts of intratumoral virus with theROD-modified vector. These results demonstrate that the tumor-specificregulation of adenoviral genes, such as E4, allows replication in vivoin permissive tumors and also demonstrates the therapeutic advantage ofthe RGD modification for a replicative adenovirus.

Local-Regional and Disseminated Models

A murine model for ovarian cancer and liver metastases of colon cancerhas been developed. These models have been useful in demonstrating theutility of the RGD modification for non-replicative adenoviral vectors,and therefore, are used herein in the context of replicativeadenoviruses containing tumor-specific promoters. The ovarian cancermodel is a local-regional model that uses the human ovarian cancer cellline, SKOV3.ipl. As these cells express SLPI, this model is useful toevaluate viruses in which the E4 or E2 gene is regulated by the SLPIpromoter. This cell line has been serially passaged in SCID mice andselected for its ability to grow aggressively in the peritoneum. FemaleSCID mice receive an i.p. injection of 2×10⁷ cells in 0.5 ml ofserum-free medium. Five days after injection, tumors start to form atthe peritoneum surface and the progression of the disease mimics thehuman disease. One week after injection, the viruses (RGD-modified orthe unmodified control) will be injected i.p. in a volume of 100 μl. Thetherapeutic viruses are also intravenously injected. Virus dosages rangefrom 10⁴ pfus to 10⁸ pfus. The therapeutic effect is measured bysurviving cells. The amount of replicating virus is measured inperitoneal lavages in time course experiments.

The model of colon cancer liver metastases uses LS174T human coloncancer cells and allows for expression of genes under the CEA promoter.In a surgical operation, cells (5×10⁸) are injected along the long axisof the spleen. Five minutes after the injection, the splenic vessels aretied off and the spleen is cut and removed. After the abdominal wall andskin are sutured, extensive liver metastases form in 7-10 days. Tailvein injection of RGD-modified and unmodified replicative adenovirusesto demonstrate systemic treatment using this model. Liver metastases arecounted in a time course experiment after virus injection.

These experiments provide in vivo data demonstrating selectivereplication and oncolytic potency of replicative vectors with restrictedreplication and enhanced infectivity. The RGD modification in the fiberof replicative adenoviruses, along with tumor-selective expression of E4or E2 in addition to E1, increases the virus' propagation efficacy andultimately its therapeutic efficacy.

Example 13 VEGF Promoter-Based Conditionally Replicative Adenovirus

In this example, the inventors exploited the expression of vascularendothelial growth factor (VEGF) in tumors for therapeutic advantage.Several studies have shown that angiogenesis is one of the key controlfactors in the growth, progression, and metastasis of solid tumors.Among the many known angiogenic factors, such as bFGF, angiogenin, IL-8,PD-ECGF, VEGF is now believed to play a pivotal role in tumor-associatedangiogenesis in a number of solid tumors.

A conditionally replication-competent adenovirus (CRAd) was constructedin which the expression of the adenoviral E1 gene was controlled by thehuman VEGF promoter. This virus achieved high levels of viralreplication in lung cancer cells and induced a substantial anti-tumoreffect in vitro and in vivo. Further enhancement of the anti-cancer cellkilling effect was achieved with tropism modification of the virus viaserotype chimerism of the adenoviral fiber knob. Theseinfectivity-enhanced VEGF promoter-based CRAds also showed a significantcell killing effect for various types of cancer cells other than lungcancer. In this regard, a dysregulated VEGF axis is characteristic ofmany carcinomas. On this basis, this current CRAd agent may be useful asa “pan-carcinoma” therapeutic agent.

Cell Culture

The NCI-H82, NCI-H460, NCI-H157, NCI-H322, NCI-H522, NCI-H1299,NCI-H358, NCI-N417, A427, A549 lung cancer cell lines; BEAS-2B, normalhuman bronchial epithelial cell line; Panc-I, pancreas cancer cell line;and HEK293 adenoviral transformed human embryonic kidney cell line wereobtained from ATCC (American Type Culture Collection, Manassas, Va.).QG56 and QG90 were provided by National Kyushu Cancer Center, Fukuoka,Japan. Human ovarian adenocarcinoma cell line SKOV3.ipl was obtainedfrom Dr. Janet Price (M.D. Anderson Cancer Center, Houston, Tex.). TheMeWo cell line was obtained from Dr. Ian R. Hart (St. Thomas Hospital,London, UK). Cells were cultured in the media recommended by eachprovider and incubated at 37° C. and 5% CO₂.

Adenovirus Vectors

The recombinant adenoviral vectors that express firefly luciferase wereconstructed through homologous recombination in Esherichia coli usingthe AdEasy system (He et al., 1998). The 2.6 kb human VEGF promoterregion derived from pVEGF-kpnl (Forsythe et al., 1996) was placed infront of the firefly luciferase gene in an Ad E1 shuttle vector,recombined with the E1- and E3-deleted adenoviral backbone vectorpAdEasy 1, then transfected into 293 cells by standard techniques toform Ad5VEGFLuc. The luciferase gene and simian virus 40-polyadenylationsignal were derived from pGL3 Basic (Promega, Madison, Wis.). As acontrol, a vector containing the ubiquitously active cytomegalovirus(CMV) immediate early promoter (derived from plasmid pCEP4; Invitrogen,Carlsbad, Calif.) instead of the VEGF promoter was also constructed andnamed Ad5CMVLuc.

The replication competent adenovirus, Ad5VEGFE1 was also generated fromthe same E1- and E3-deleted adenoviral backbone vector. Briefly, thefragment corresponding 489 bp to 3533 bp from the left end of the type 5adenoviral genome was amplified by PCR and inserted in the E1 deletedregion of the backbone vector. This fragment contains thetranscriptional start site of the E1A gene but not the native E1Apromoter. The 2.6 kb VEGF promoter region was placed upstream of thisfragment. A control vector was also constructed in which the CMVpromoter was placed in the same position upstream of E1A. The strategyfor these constructs is summarized in FIG. 17. Fiber modified CRAd,Ad5/3VEGFE1 was generated in similar manner as Ad5VEGFE1 but usingAd5/3E1-E3-deleted backbone vector derived from Ad5/3lucl containing Ad3knob in place of Ad5 wild-type knob gene as described previously(Krasnykh et al., 1996). To compare the differences in infectivitybetween the Ad5 and Ad5/3 chimeric vectors on the target cells, an Advector (Ad5/3lucl) that contains a CMV driven luciferase gene in E1 wascompared to AdCMVLuc. Wild type p53 protein expressing adenovirus,Ad5p53 which contains CMV driven p53 cDNA was provided from Dr. Ueno(University of Occupational and Environmental Health, Kitakyusyu, Japan)(Takayama et al., 1998)

The viruses were propagated in the adenovirus packaging cell line,293HEK, and purified by double CsC1 density gradient centrifugation,followed by dialysis against phosphate-buffered saline with 10%glycerol. The viral particle (VP) concentration was determinedspectrophotometrically, using a conversion factor of 1.1×10¹² viralparticles per absorbance unit at 260 nm, and standard plaque assays on293 cells were performed to determine infectious particles.

Analysis of VEGF RNA Expression

The VEGF RNA status of cell lines was analyzed by reverse transcriptionand polymerase chain reaction (RT-PCR) as described previously (Ohta etal., 1996). Total cellular RNA was extracted from 1×10⁷ cells using theRNeasy kit (Qiagen, Valencia, Calif.) and analyzed for VEGF andglyceraldehydes-3-phosphate dehydrogenase (GAPDH) RNA with the GeneAmpRNA PCR core kit (Applied Biosystems) as described by manufacturer.Briefly, 500 ng of total RNA was reverse-transcribed with the randomhexamer and murine leukemia virus reverse transcriptase (50° C., 30 min)and amplified by PCR with 50 nM of primer pairs described below using acycling program (initial step of 95° C. for 15 min, 27 cycles of 95° C.for 1 min and 60° C. for 1 min and 72° C. for 1 min, final step of 72°C. for 10 min). The primers used for the analyses were as follows: VEGFsense, 5′GAAGTGGTGAAGTTCATGGATGTC3′, SEQ ID NO:6; VEGF antisense,5′CGATCGTTCTGTATCAGTCTTTCC3′, SEQ ID NO:7; GAPDH sense,5′CCTTCATTGACCTCAACTA3′, SEQ ID NO:8; GAPDH antisense,5′GGAAGGCCATGCCAGTGAGC3′, SEQ ID NO:9.

Measurement of VEGF Protein in Culture Media

The VEGF protein expression was evaluated as described previously.Briefly, 1×10⁵ cancer cells were cultured for 24 h in serum free media,and then the medium was collected. After centrifugation, the supernatantwas stored at −80° C. until the assay. The VEGF protein in the culturemedium was determined using an ELISA kit (Quantikine Human VEGFImmunoassay, R&D Systems, Minneapolis, Minn.) according to themanufacturer's instructions. Each of the values given here is the meanof triplicate determination with respect to standardized cell numbers,1×10⁵ cells.

In Vitro Analysis of VEGF Promoter Activation

The activity of the VEGF promoter in an adenovirus context was analyzedby infection of cells with luciferase expression vectors as reportedpreviously (Adachi et al., 2001). Briefly, cells were plated in 12-wellplates in triplicate at a density of 1×10⁵ cells/well. The next day, thecells were infected with Ad5VEGFLuc or Ad5CMVLuc at a MOI of 10 pfu/cellin DMEM with 2% FCS for 3 h and then maintained in complete medium. Theinfected cells were harvested and treated with 100 ml of lysis buffer(Promega, cat #E153A) after 2 days culture. A luciferase assay(Luciferase Assay System; Promega) and a FB12 luminometer (Zyluccorporation) were used for the evaluation of luciferase activities ofAd-infected cells. Luciferase activities were normalized by the proteinconcentration in cell lysate (Bio-Rad DC Protein Assay kit).

In Vivo Analysis of VEGF Promoter Activation

For determination of luciferase gene expression in mouse organs, nudemice (Charles Rivers) received 1×10⁹ pfu of Ad5CMVluc or Ad5VEGFLuc bytail vein injection as described previously (Adachi et al., 2001). Twodays later, mice were sacrificed, and the livers, kidneys, lungs,spleens were resected to measure the luciferase gene expression. Theresected organs were placed in the polypropylene tubes, and immediatelyfrozen in ethanol/dry ice. Frozen tissues ground to a fine powder waslysed using a tissue lysis buffer (Promega), and then luciferaseactivity was determined using a luciferase assay kit (Promega). Theluciferase activity was normalized by protein concentration in thetissue lysate.

Analysis of Viral Genome Amplification

Viral DNA amplification was assessed as reported previously (Adachi etal., 2001). Cells were plated in a 12-well culture plate in triplicateat the density of 1×10⁵ cells/well. After overnight culture, cells wereinfected with replication-competent Ads (Ad5VEGFE1, Ad5CMVE1 or Ad5 wt)or non-replicative Ad (Ad5CMVLuc) at the MOI of 10 for 3 h and thencultured for 24 h. The harvest of infected cells was followed by viralDNA isolation using Blood DNA kit (Qiagen, Valencia, Calif.). Viral DNAwas eluted with 100 ml of elution buffer [10 mM TrisCl (pH 8.5)]. Elutedsamples (1 ml) were analyzed by real-time PCR analysis to evaluateAdenoviral E4 copy number using a LightCycler (Roche). Oligonucleotidescorresponding to the sense strand of Ad E4 region(5′-TGACACGCATACTCGGAGCTA-3′, 34885-34905 nt, SEQ ID NO:10), theantisense strand of E4 region (5′-TTTGAGCAGCACCTTGCATT-3′, 34977-34958nt, SEQ ID NO:11), and a probe (5′-CGCCGCCCATGCAACAAGCTT-3′, 34930-34951nt, SEQ ID NO:12) were used as primers and probe for real-time PCRanalysis. The PCR conditions were as follows: 35 cycles of denaturation(94° C., 20 s), annealing (55° C., 20 s), and extension (72° C., 30 s).Adenovirus backbone vector pTG3602 (Chartier; Transgene, Strasbourg,France) was available for making a standard curve for Ad E4 DNA copynumber. E4 copy numbers were normalized by the b-actin DNA copy number.

In Vitro Cytotoxicity Assay

For determination of virus-mediated cytotoxicity, 5×10³ cells wereplated in 96-well plates in triplicate. After overnight culture, cellswere infected with each Ads at various MOI for 3 h. The infection mediumwas then replaced with RPMI1640 containing 10% FCS. Viable cells usingMTS assay (CellTiter 96 Aqueous Non-Radioactive Cell ProliferationAssay; Promega) were evaluated every 3 days. The MTS color developmentwas quantified as optical density at 490 nm by an EL 800 UniversalMicroplate Reader (Biotec Instruments Inc.)

To visualize the cytotoxic effect, crystal violet staining was alsoperformed. Cells (2×10⁵) were plated in 12-well plates and infected witheach Ad at various MOI for 3 h. The infection medium was replaced withgrowth medium the next day. When cell lysis was observed, cells werefixed and stained with 1% crystal violet in 70% ethanol for 45 min,followed by washing with tap water to remove excess color. The plateswere dried, and images were captured with a Kodak DC260 digital camera(Eastman Kodak, RochestertNY). All experiments were performed induplicate wells.

In Vivo Studies—Tumor Formation in Nude Mice

Tumor suppressive effect in vivo was analyzed as described previously(Takayama et al., 2000). Briefly, H157 cells (5×10⁶) were injected s.c.into the dorsal skin of nude mice, and tumor growth was monitored for 25days. Tumor volume was calculated according to the formula a²×b, where aand b are the smallest and largest diameters, respectively as describedpreviously. When tumor formation was seen 10 days after inoculation,1×10⁸ pfu of each virus was injected into the tumor directly. Student'st test was used to compare tumor volumes, with p<0.05 being consideredsignificant.

VEGF mRNA and Protein Expression in Various Cell Lines

The inventors first investigated a panel of twelve non-small cell lungcancer cell lines, one bronchial epithelial cell line (BEAS-2B) as anormal cell control, one ovarian cancer cell line (SKOV3.ipl), onegastric cancer cell line (MKN28), and a pancreatic cancer cell line(Panc-I) for VEGF mRNA expression using a RT-PCR method. In this regard,there are four structural variants of VEGF (VEGF121, VEGF165, VEGF189,and VEGF206) resulting from alternative mRNA splicing in the regionsencoding the cytoplasmic domains. FIG. 18A shows amplification of a 408bp fragment (representing VEGF121 cDNA) and a 541 bp fragment(representing VEGF165 cDNA) in all cell lines tested. The intensity ofeach band (VEGF121 and VEGF165) was similar in all cancer cells tested.The PCR bands corresponding to VEGF189 (615 bp) and VEGF206 (666 bp)were minimal or not detected, indicating VEGF121 and VEGF165 were thedominant isoforms in these cell lines. These results are consistent withthose of previous similar studies of primary lung cancer tissues. Of thecells tested, H157, A427, N417, H358 and SKOV3.ipl showed relativelyhigh expression of VEGF mRNA, while the control normal cell line BEAS-2Bshowed a less intense band than the cancer cell lines, although the bandcorresponding to VEGF121 was detected at very low levels.

The correlation between mRNA expression and protein expression for VEGFwas also investigated. As shown in FIG. 18B, the VEGF protein expressionlevels also varied between cell lines. H157 secreted the highest amountof VEGF protein into the culture media, and the concentration was over100 times higher than that of BEAS-2B. Comparison between FIGS. 18A and18B revealed that the VEGF mRNA expression level positively correlatedwith VEGF protein expression level. These results thus suggested theVEGF promoter activity can be predicated from the VEGF proteinconcentration of tumor cellular substrates.

Transgene Expression by VEGF Promoter in the Ad Context In Vitro

Candidate tumor-specific promoters may lose their specificity whenplaced in the context of the Ad genome. Thus, the VEGF promoter activitywas assessed in an Ad vector (Ad5VEGFluc) containing the luciferase geneas a reporter. This was examined in several cell lines that representedthe range of VEGF levels detected in FIG. 18. In all of the cells linestested, luciferase expression was achieved using the positive controlAd5CMVLuc, which contains the luciferase gene driven by thenon-selective viral CMV promoter. These results demonstrate that theA247 and H157 cells were most susceptible to Ad5 infection, exhibitingluciferase levels over 100 times higher than these of H460 as shown inupper panel of FIG. 19. To standardize the differential susceptibilityto Ad5 infection between cell lines, VEGF promoter activity is thusshown as the percentage of luciferase activity of Ad5VEGFLuc relative toAd5CMVLuc. As shown in the lower panel in FIG. 19, H157 cells showed thestrongest VEGF promoter activity which was 28% of CMV promoter activity.In contrast, BEAS-2B cells, which presented the lowest VEGF promoteractivity, was less than 0.1% of CMV. This low transgene expression seenwith the VEGF promoter in the adenoviral context with BEAS-2B wasconsistent with other recent reports. Other cell lines demonstratedvarious VEGF promoter activities which correlated with the mRNAexpression level for each cell lines tested (FIG. 18A). Based on thesedata, it is concluded that the VEGF promoter was able to inducetransgene expression in VEGF producing cells and, importantly, that thepromoter retained its specificity when configured in the Ad genomecontext.

Transgene Expression by VEGF Promoter in the Ad Context In Vivo

A key limitation of adenovirus-mediated cancer gene therapy is thepotential for toxicity to non-target organs. Because Ad exhibits amarked tropism for the liver, it is important to determine whether theVEGF promoter would have low activity in the liver in vivo. Such a“liver off” phenotype would be critical to avoid any toxic effects ofVEGF promoter CRAd therapy. Normal liver was reported to exhibit minimalVEGF expression.

On this basis, Ad5VEGFLuc or Ad5CMVLuc (as a positive control) wereinjected i.v. via the tail vein into mice and the level of transgeneexpression at day 2 was determined (FIG. 20). In this assay, transgeneexpression in the liver induced by the VEGF promoter was a mean 270-foldless than that seen with the CMV promoter. These results thus confirmthe key property of VEGF promoter fidelity in vivo in the context of theAd vector used. Furthermore, the “liver off” phenotype of the VEGFpromoter makes the use of a VEGF promoter CRAd feasible in a systemicdelivery context.

VEGF Promoter Driven CRAd Shows Replication Specificity

To exploit the cell specificity of the VEGF promoter in a CRAd context,the inventors then constructed a recombinant Ad (Ad5VEGFE1) in which thenative E1 promoter was replaced with the 2.6 kb human VEGF promoter. Thegenomic structures of replication competent Ads used in this study aredepicted in FIG. 17. An Ad in which E1 expression is controlled by thenon-selective viral CMV (Ad5CMVE1) promoter was used as control. Theseviruses are deleted in the E3 region to accommodate the large VEGFpromoter and the E1A gene region. The deleted E1A promoter region,containing the native E1A TATA box, was replaced with either the VEGFpromoter or CMV enhancer/promoter to produce the viruses Ad5VEGFE1 orAd5CMVE1, respectively.

To determine the specificity of replication of the AdVEGFE1, the highVEGF expressing cell line (H157) and low expressing cell line (BEAS-2B)were infected with the Ad vectors, and then quantitative real-time PCRwas used to determine the level of amplification of viral DNA. Thenon-replicative Ad5CMVLuc and wild-type Ad5 virus (Ad5 wt) were used asnegative and positive controls, respectively. Since all viruses testedcontained the Ad E4 region, viral DNA was quantified by E4 copy numbervia real-time PCR. As shown in the upper panel of FIG. 21. the Ad5VEGFE1viral genome replicated in the high VEGF producing cancer cells H157 toa similar extent as did the Ad5CMVE1 genome. The nonreplicativeAd5CMVLuc showed a background level of E4 signal, indicating noreplication in this cell line. Importantly the replicative capacity ofAd5VEGFE1 decreased in the low VEGF expressing BEAS-2B cells, withvalues 3-logs lower than that for Ad5CMVE1 (lower panel in FIG. 21).These results indicate that the VEGF promoter retains fidelity in thereplication competent adenoviral context and mediates tumor-specificadenoviral replication.

Specific Cell Killing Efficacy of VEGF Promoter-Driven CRAd

The ability of Ad5VEGFE1 to achieve cell killing in the VEGF-positivecell lines was determined using a MTS assay. The viability of the highVEGF expressing H157 cells and the low VEGF expressing BEAS-2B cells wasquantified every three days after virus infection as shown in FIG. 21A.For the H157 cells. Ad5VEGFE1 showed cytotoxic effect as strong as thatof the Ad5CMVE1 positive control virus. All cancer cells were killed byday 9 with infection at a low MOI. The relatively steep fall in thesurvival curve after day 5 suggested a minimal temporal requirementbefore sufficient replication occurred to induce toxicity.

To reconcile these results with an alternative gene-based approach tocancer treatment which has been proposed, the Ad5VEGFE1 cytotoxic effectwas compared with that of Ad5p53, which encodes the wild-type p53 geneand has been employed in human clinical trials. In this regard, it haspreviously been shown that H157 cells, which have a mutated p53 gene,undergo apoptosis when infected with Ad5p53. Ad5p53 infection of H157cells at MOI 0.1 showed a weak cytotoxic effect compared with Ad5VEGFE1.Similar results were obtained with A427 cells (data not shown). Incontrast to the effect in the cancer cells, BEAS2B cells were resistantto Ad5VEGFE1 toxicity even with infection at a high MOI of 10. Thesedata were consistent with the crystal violet staining appearance asshown in FIG. 21B.

Tumor Growth Suppression by Ad5VEGFE1 In Vivo

The inventors next investigated whether Ad5VEGFE1 could suppress tumorgrowth in vivo. To this end, subcutaneous tumors established in nudemice were directly injected with either Ad5CMVLuc, Ad5VEGFEI or Ad5p53.Tumors become visible and injectable 10 days after subcutaneousinoculation. Previous work revealed that the inoculated H157 cells havecompleted angiogenesis at this time, and in this regard resembleadvanced human tumors (Takayama et al., 2000). For these studies, 1×10⁸pfu of each virus was injected into the tumor directly and each tumorwas observed for 2 weeks. As shown in FIG. 23, tumor injected withAd5CMVLuc increased in size. Ad5p53 suppressed tumor growth partially;however, the suppressive effect was minimal. In contrast, Ad5VEGFE1suppressed the tumor growth to a significantly greater degree thanAd5p53. These findings suggested that CRAd may be a more efficaciousagent than non-replicative virus-based gene therapy approaches such asAd5p53.

Improvement of CRAd Potency Via Fiber Modification

The oncolytic effect of any CRAd is dependant on the infectivity of thecancer cells as well as promoter activation specificity. Based on theseconcepts, the inventors endeavored to achieve improvement of adenovirusinfectivity as a means to enhance the anticancer effect achieved via theCRAd agent. In this study, it is noted that adenovirus infectivity forH460 lung cancer cells and SKOV3.ipl ovarian cancer cells was almost 2orders of magnitude lower than that of H157 and A427 lung cancer cells(FIG. 19). This differential infectivity is likely the basis ofdifferential CRAd efficacy noted in these contexts. In this regard, ithas been previously reported that infectivity of serotype 5 adenoviruscan be improved by fiber modifications. For example, a modifiedadenovirus with a chimeric fiber which expresses Ad3 knob instead of Ad5knob. (Ad5/3) showed enhanced infectivity for various tumor cells thatwas otherwise Ad refractory.

The inventors therefore analyzed the effect of infectivity enhancementvia knob serotype chimerism for the cell lines tested in this study. Asshown in FIG. 24, the luciferase activities with the Ad5/3 vectorincreased in all 6 cell lines tested. The increases observed werebetween 5.1 times in Panc I cells and 39.4 times in A549 cells. Thesefindings led the inventors to construct an Ad5/3VEGFE1 in which the Ad5knob is replaced with Ad3 knob. Ad5/3VEGFE1 was generated and propagatedas described in Materials and Methods. The oncolytic effect ofAd5/3VEGFE1 relative to Ad5VEGFE1 for the various cancer cells wasevaluated using infection at 1 MOI (FIG. 25). Cytopathic effect withAd5/3VEGFE1 infection was seen rapidly. almost 2 days earlier than thatwith Ad5VEGFE1 in all cell lines. In this experiment, complete celldeath was seen for all lines infected with Ad5/3VEGFE1 nine days afterinfection whereas a significant number of cells survived with Ad5VEGFE1infection. Moreover. Ad5/3VEGFE1 showed a stronger cell killing effectfor H322 cells and SKOV3.ipl cells compared with Ad5CMVE1. These resultssuggested that infectivity enhancement with modified adenovirus fibercould improve the cell killing effect of the VEGF promoter CRAd.

Conditionally replicative adenovirus (CRAd) represents a promising newtherapeutic approach for malignancies resistant to conventionaltreatments. The current example demonstrates a strategy based on the useof a replication-competent Ad controlled by a VEGF promoter.Furthermore, it is demonstrated that AdVFEGFE1 is applicable for thetreatment of a wide spectrum of tumors. With regard to gene therapy oflung cancer, replication incompetent Ad expressing wild-type p53 iscurrently being employed in human clinical trials. Whilereplication-incompetent viral vectors have demonstrated great promise asanticancer agents in preclinical studies, this has not been translatedinto patient benefit in the clinical setting. The poor anticancer effectwith replication-incompetent Ad is partly due to limited penetration ofthe vector into the tumor mass. In this regard, CRAd agents are designedto achieve intratumoral spread and penetration by virtue of theirreplicative capacity.

For clinical application, prevention of hepatic toxicity by adenoviralagents is an important consideration. Tumor cells infected withreplication-competent Ad may release new viruses in vivo. Suchdissemination could predicate treatment related toxicity, especially inthe context of the liver as this is the predominant site of Ad vectorlocalization after systemic injection. In this regard, the presentexample shows that the VEGF promoter exhibits extremely limited promoteractivity in the liver and thus may avoid untoward hepatic injury. SinceAdVEGFE1 exhibited a high degree of specificity in both replication andcytotoxicity which correlated with target cell VEGF expression, it wouldbe predicated to be less toxic to the liver compared with AdCMVE1 orwild-type Ad. Results of a phase I clinical trial with VEGF inhibitorsshowed that these agents were well tolerated, indicating a marginal rolefor VEGF signaling in normal organs under physiological conditionsexcept the ovary during the menstrual cycle.

An emerging strategy for cancer therapy is the use of conditionallyreplicative adenoviruses (CRAds) that are designed to exploit keydifferences between tumor cells and normal cells to allowtumor-selective viral replication and oncolysis. Two basic strategieshave been employed to generate CRAds. A type I approach, such asAd-dl1520 (ONYX-015) or AdD24, involves directly mutating Ad genes suchas E1 to take advantage of the disordered cell cycle regulation in tumorcells with functionally deficient p53 or RB signaling, respectively. Thetype II approach involves replacement of wild-type Ad promoters withtumor-specific promoters to drive the expression of genes essential forAd replication.

A consideration for the clinical employment of type II CRAd is that therelevant promoter activity in each tumor should be confirmed beforetreatment. In this regard, it is clear that tumors with low promoteractivity are resistant to type II CRAds containing that promoter.Therefore it is important to evaluate the promoter activity a priori toavoid potentially non-indicated therapy. Analysis for RNA statusrequires tissue obtained from the patient to prepare RNA samples forRT-PCR or northern blotting. Precise evaluation of promoter activitywith a reporter gene such as luciferase is more difficult in theclinical setting generally. Considered in this context, it is clear thatthe VEGF promoter has an advantage for its activity evaluation. Resultsin FIGS. 18 and 19 demonstrated that there is a positive correlationbetween VEGF mRNA expression level, VEGF protein expression level, andtransgene activation by the VEGF promoter. Taken together these datasuggest that the VEGF promoter activity within a tumor can be predictedfrom VEGF protein expression levels. Of note, VEGF protein is easilydetectable in clinical samples by ELISA evaluation of fluid samples andimmunohistochemical staining of tissue samples. Thus these tests canpotentially be employed to prospectively select the most appropriatepatients for consideration of VEGF promoter CRAd therapy in the clinicalsetting.

VEGF production is an important mechanism for the development oftumor-associated angiogenesis in many types of tumors. In fact, manytypes of cancer are already known to express VEGF protein at significantlevels and this VEGF expression is associated with poor prognosis inseveral disease contexts including leukemia, breast cancer, colorectalcancer, hepatocellular carcinoma, ovarian cancer and non-small cell lungcancer. It appears that more advanced stage tumors actually expresshigher levels of VEGF protein. Of note, VEGF gene expression is known tobe regulated transcriptionally. Although several transcription factorsbind to the cis-elements on the promoter, hypoxia inducible factor (HIF)is the key factor for activation of the promoter. In this regard, thecentral regions of tumors are often hypoxic and necrotic due todecreased blood flow. Immunohistochemical analysis of primary tumorsamples shows that VEGF protein expression is enhanced in the tumortissue adjacent to necrotic regions. On the other hand, some types ofcancer are known to express the HIF protein constitutively despite theoxygen tension, leading to an increase VEGF promoter activation. Takentogether these findings suggest that the antitumor effect of AdVEGFE1may be even more efficacious in large in vivo tumors than under thenormoxic conditions under which the above in vitro experiments wereperformed.

The cell killing effect of a type II CRAd may be improved by severalmechanisms such as promoter induction, infectivity enhancement, or anarmed CRAd strategy. A major obstacle to be overcome in Ad5-based cancergene therapy has been the paucity of the primary receptor, CAR, whichfrequently characterizes human primary tumor cells. Furthermore, downregulation of CAR may be associated with a more malignant phenotype. Dueto variable expression of CAR on human primary cancer cells, the utilityof Ad5 as a cancer gene therapy vector may be compromised, limiting theoverall efficacy of any Ad-based cancer gene therapy, including the useof CRAds agents. On this basis, approaches to circumventtumor-associated CAR deficiency are required. In this regard, the nativeAd5 tropism can be modified to enhance Ad infectivity. One approach ispseudotyping, i.e., retargeting Ad by creating chimeric fiberspossessing knob domains derived from alternate serotypes which bind toreceptors other than CAR. To this end, nonreplicating Ads containingchimeric fibers with the tail and shaft domains of Ad serotype 5 and theknob domain of serotype 3 have been constructed (Krasnykh et al., 1996).Previous work has revealed that a distinct Ad3 receptor exists inovarian cancer cells, and that the Ad5/3 chimeric vector is retargetedto the Ad3 receptor. Based on these findings, a CRAd exploiting theAd5/3 chimeric approach was constructed in this study. Results presentedabove indicate that Ad5/3VEGFE1 showed a stronger cell killing effectthan that of the Ad5-based CRAd, likely on this basis of conferredinfectivity enhancement.

In conclusion, the data presented here provide a basis for theadvancement of replication-competent adenovirus strategies based on theVEGF promoter for the therapy of various cancers. Furthermore, a CRAdbased on the Ad5/3 chimeric vector is a promising way to enhance theanti-tumor potency via infectivity enhancement for cancer cells. Giventhe relevance of a dysregulated VEGF axis in a broad spectrum of tumortypes, as well as the frequency of deficient adenoviral receptor CAR inthe context of epithelial neoplasms, the current infectivity enhancedVEGF promoter CRAd may represent a “pan-carcinoma” CRAd with broadpotential utilities.

Example 14 CXCR-4 or Survivin Promoter-Based Conditionally ReplicativeAdenovirus

The enormous promise of CRAd vectors for cancer gene therapy has beenestablished and has resulted in the rapid clinical translation of thisapproach. The present example provides a CAR-independent vector that wasrendered selectively replicative via the CXCR4 or survivin promoter.These vectors have improved transductional efficiency and specificityrequired for human clinical trials and allow full realization of thepotential benefits of the CRAd approach for breast cancer.

Derivation of a Novel, CAR-Independent Ad Vector

Many clinically relevant tissues are refractory to Ad5 infection due tonegligible CAR levels. Some non-human Ads display CAR-independentinfection of human cells. Canine adenovirus type 2 (CAd2) infects humancells via CAR, but also displays CAR-independent infection ofCAR-negative human cells with identical entry kinetics to Ad5 (Soudaiset al., 2000). To create an Ad vector for infection of CAR-deficientcells, a “knob-switching” technology (Krasnykh et al., 1996) wasemployed to engineer a non-replicative, Ela-deleted Ad vector,AdCK/CMV-Luc, which contains the knob domain of the canine adenovirustype 2 (CAd2) and a luciferase reporter gene. The AdCK/CMV-Luc vectorwas rescued in HEK 293 cells, and the correct chimeric fiber DNAsequence was confirmed. This novel Ad was propagated in HEK 293 cells,and was grown to high titers and purified by traditional methods.

To confirm that AdCK/CMV-Luc displays CAR-independent infection,infection assays were performed on human glioma cells, U118-CAR that wasengineered to express human CAR, and the parental CAR-negative U118cells. In CAR-negative U118 cells, AdCK/CMV-Luc showed 15-fold higherluciferase activity than the isogenic control, Ad5/CMV-Luc (FIG. 26).Furthermore, in U118-CAR cells, AdCK/CMV-Luc had similar luciferaseactivity to Ad5/CMV-Luc. Importantly, addition of excess recombinant Ad5knob protein blocked Ad5/CMV-Luc infection, but not that ofAdCK/CMV-Luc, indicating AdCK/CMV-Luc has novel, CAR-independenttropism. In addition, AdCK/CMV-Luc demonstrated a 10-fold infectivityenhancement in ovarian SKOV3.ipl vs. Ad5/CMV-Luc (data not shown).

Initial Characterization of Potential Breast Cancer-Selective Promoters

The inventors have obtained full length human CXCR4 and survivinpromoters for evaluation as breast cancer-specific promoters for CRAdagents. CXCR4, identified as co-receptor for HIV-1, is also a chemokinereceptor recently implicated in the metastatic homing of breast cancercells to alternate tissues. CXCR4 gene expression is markedlyupregulated in breast cancer cells, but is undetectable in normalmammary primary epithelial and stromal cells. Expression of survivin, amember of the inhibitor of apoptosis (IAP) family, is associated withloss of apoptosis in breast cancer, and is a significant prognosticparameter of poor outcome. Over 70% of stage I to IH breast carcinomashave been shown to express survivin, with undetectable expression levelsin adjacent normal differentiated tissues or stromal cells.

To verify the overexpression of CXCR4 and survivin in breast cancercells, real-time PCR mRNA analysis was performed in two breast cancercell lines, DU4475 and MDA-MB-361 (FIG. 27). Both cell lines showedelevated survivin and very high CXCR4 mRNA levels compared with humanfibroblast (HFB), prostate (BxPC-3 and AsPC-1) or ovarian (OV4 andSKOV3.ipl) cancer cells. To evaluate promoter specificity in the Adgenome context, a panel of non-replicative Ad vectors was constructedwith a luciferase reporter gene under the transcriptional control of theCXCR4 (Ad5/CXCR4-Luc) and survivin (Ad5/survivin-Luc) promoters (FIG.28). As expected, luciferase activities were elevated in MDA-MB-361breast cancer cells, expressed as percent of Ad5/CMV-Luc (FIG. 29).

Evaluation of CAR-Independent Breast Cancer CRAd Agents For ImprovedOncolytic Potency

Preliminary data clearly show that the CAR-independent vector,AdCK/CMV-Luc, provides substantial infectivity enhancement forCAR-deficient substrates. The CAd2 knob domain can be incorporated intothe fiber of the CXCR4 and survivin CRAds via well establishedrecombinational strategies. The viral replication and oncolytic cellskilling activities of the newly derived CAR-independent CRAds can becompared to their wild-type counterparts as follows. Various doses ofCRAds are added to target cells in culture. At various time points,cells are evaluated for CRAd replication using automated PCR-based assaybased on the TaqMan approach. Crystal violet staining of infectedplates, plus MTT/XTT viability assays can be used to provide indices ofreplication-induced oncolysis. Various input m.o.i's will be evaluatedand dose equivalencies established. The CAR-independent CRAds shouldachieve increased oncolysis at lower m.o.i., indicating their increasedpotency. As an additional assay, the inventors will employ a spheroidcell culture system that provides growth of cell lines and primarycultures in a three-dimensional configuration. This novel system allowsdetermination of efficacy of agents that operate via “amplification”,such as CRAds. These studies will determine the relevance of infectivityenhancement for the context of breast cancer CRAds. CRAd agents thatprovide breast cancer-specific replication as well as increased breastcancer infectivity will allow early definition of a lead agent forfurther pre-clinical development

Analysis of Therapeutic Utility in Murine Model Systems

Studies accomplished to this point will establish a “lead agent” forfurther evaluation. The use of a murine xenograft model system willprovide a means to determine the therapeutic utility of this agent. Fortherapeutic analysis studies, SCID mice are xenotransplantedsubcutaneously with human breast cell lines, including MDA-MB-231 cells.This model will be challenged with the CRAd agent via distinct routes:intratumoral, intraperitoneal and intravenous. The former routeparallels treatment of loco-regional disease via CRAd delivery. Thelatter route parallels systemic delivery relevant to disseminateddisease. Tumors will be harvested post-treatment and assayed for CRAdreplication via TaqMan PCR. Direct three-dimensional measurement oftumor regression can be performed as a function of time and viral dose.Control agents include non-replicative Ad, as well as replicativewild-type Ad. Comparisons are also made between the CAR-independentCRAd, and its native-tropism counterpart, and control Ads. Ectopiclocalization of CRAd occurs largely in liver. Thus, this organ providesthe best index of CRAd-induced toxicity. Therefore, treated animals willundergo histopathological analysis for evidence of CRAd-relatedpathology. These efficacy and toxicity studies will provide directinsight into the therapeutic index of these CRAd agents, and predict thepre-clinical/clinical pathway for a human breast cancer clinical trialwith the novel CRAd agents.

Example 15 Uses of Survivin Promoter in Double Targeting to OvarianCarcinoma

This example discloses double targeting for ovarian cancer cells invitro and in vivo that involves transductional targeting andtranscriptional targeting. Transductional targeting is achieved byretargeting adenoviral vector to tumor-specific cell surface markers,such as epidermal growth factor receptor (EGFR), by a bi-functionaladaptor or modified fiber of adenoviral vector. Transcriptionaltargeting can enhance tumor specificity by using a tumor-specificpromoter (such as surviving promoter) to restrict transgene expressionto tumor cells. It is anticipated that ovarian tumor specificity will beenhanced by targeting based on a tumor specific promoter, survivin, anda retargeting site, EGFR, which is overexpressed in ovarian cancer; andthe toxicity to normal tissue will be limited by enhancing tumorspecificity and decreasing the dose of administration.

Transductional Targeting by EGFR-Retargeted Adenoviral Vector

Several human ovarian cell lines have been chosen for this study. Todetermine EGF receptor expression on cell surface by flow cytometry,cells (10⁴) are sorted on FACScan flow cytometry after treated with1^(st) antibody (5 mg/ml) mAb 425, a monoclonal antibody anti-EGFR and2^(nd) antibody (5 mg/ml), a goat anti-mouse IgG labeled with FITC.

Adenoviral vector will be targeted to EGF receptor by using a fusionprotein sCAR-EGF encoded by the construct pFBsCAR6hEGF. The donorplasmid pFBsCAR6hEGF will be transformed into competent DH10Bac E. colicells to generate a recombinant Bacmid. The fusion protein sCAR-EGF willbe produced in High Five cells, purified with NI-NTA resin, and detectedby Western Blot.

To compare EGFR-targeted Ad gene transfer among human ovarian cancercell lines, human ovarian cancer cells (5×10⁴ cells) will be infectedwith AdGL3BCMV (M.O.I.=100) pre-incubated with various doses of thesCAR-EGF fusion protein (0, 5, 15, and 20 mg) at room temperature for 30min. Forty-eight hours post-infection, the luciferase activity will bedetermined with the luciferase Assay System on the lumicount. Acompetition test will be performed by using mAb A-431 to blockretargeting site on the surface of tumor cells.

Transcriptional Targeting Using Survivin Promoter

mRNA levels of survivin are over expressed in the ovarian cell line,SKOV3.ipl, but not in OV4, as determined by real-time PCR using aLightCycler. The results indicated survivin transcriptional activity inSKOV3 ipl was 40-fold and 10-fold higher than that of 2 control celllines, human fibroblasts and human mammary epithelial cells,respectively. The results also showed that, with in vitro analysis ofthe survivin promoter in an adenoviral context, the luciferaseactivities were 4- and 5-fold higher in SKOV3op.1 and OVCAR3 cell linesthan the 2 control cell lines, respectively. These cells were infectedwith AdGL3BSurvivin or AdGL3BCMV. AdGL3Bsurvivin is a vector in whichthe reporter gene luciferase is driven by the ovarian tumor specificpromoter survivin, whereas AdGL3BCMV is a control for normalizing theluciferase activity driven by survivin promoter (set CMV promoteractivity to 100%).

To determine survivin promoter activity in ovarian cancer cell lines invitro and in vivo, luciferase activity driven by survivin promoter aredetected as a percentage of that driven by CMV promoter in differentovarian cancer cell lines. Briefly, 5×10⁴ cells are infected withAdGL3Bsurvivin or AdGL3BCMV (M.O.I=100) in a conventional condition.Luciferase activities are measured 48 hours post-infection.

To analysis the distribution of luciferase gene expression in mouseorgans, six mice are injected with 10⁹ pfu of AdGL3B-Survivin orAdGL3BCMV via tail vein. Two days later, major organs are harvested, andluciferase activity determined.

Double Targeting for Ovarian Cancer Cells In Vitro and In Vivo

To examine double specific targeting for ovarian cancer cells in vitro,luciferase activity driven by survivin promoter will be determined as apercentage of that driven by CMV promoter in different ovarian cancercell lines and controls. Briefly, 5×10⁴ ovarian cancer cells areinfected with AdGL3BSurvivin or AdGL3BCMV (M.O.I.=100) bothpre-incubated with various amount of fusion protein sCAR-EGF (0, 5, 10,15, 20 mg) at room temperature for 30 min. As a control, sCAR6His isused for blocking the native interaction of CAR. Luciferase activitieswill be measured 48 hours post-infection.

To examine double targeting in primary ovarian cancers, the experimentdescribed above can be repeated with primary ovarian cancers obtainedfrom 4-5 patients.

To determine double specific targeting for ovarian cancer in vivo, 2×10⁷cells of SKOV3ipl will be inoculated subcutaneously into flank of BALB/cnu/nu mice. When the tumor reaches a diameter of 6-8-mm, intra-tumor ori.v. injection will be performed with 5×10⁸ pfu of AdGL3BSurvivin orAdGL3BCMV pre-incubated with suitable amount of fusion protein sCAR-EGF.Two days later, the tumor will be resected for luciferase analysis.

The following references were cited herein:

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1. An infectivity-enhanced conditionally-replicative adenovirus, whereinsaid adenovirus possesses enhanced infectivity towards a specific celltype due to a modification or replacement of the fiber of a wildtypeadenovirus, said modification or replacement results in enhancedinfectivity relative to said wildtype adenovirus, and wherein saidinfectivity-enhanced conditionally-replicative adenovirus has at leastone conditionally regulated early gene, said early gene conditionallyregulated such that replication of said infectivity-enhancedconditionally-replicative adenovirus is limited to said specific celltype.
 2. The infectivity-enhanced conditionally-replicative adenovirusof claim 1, wherein said cell type is a tumor cell.
 3. Theinfectivity-enhanced conditionally-replicative adenovirus of claim 1,wherein said modification or replacement to the fiber results incoxsackie-adenovirus receptor independent gene transfer with respect tothe type 5 receptor.
 4. The infectivity-enhancedconditionally-replicative adenovirus of claim 1, wherein saidmodification or replacement to the fiber is selected from the groupconsisting of introducing a ligand into the HI loop of said fiber,replacing said fiber with a substitute protein which presents atargeting ligand, and introducing a fiber knob domain from a differentsubtype of adenovirus.
 5. The infectivity-enhancedconditionally-replicative adenovirus of claim 4, wherein said ligand isselected from the group consisting of physiological ligands,anti-receptor antibodies and cell-specific peptides.
 6. Theinfectivity-enhanced conditionally-replicative adenovirus of claim 4,wherein said ligand comprises a tripeptide of Arg-Gly-Asp (RGD).
 7. Theinfectivity-enhanced conditionally-replicative adenovirus of claim 4,wherein said ligand comprises a peptide having the sequence CDCRGDCFC.8. The infectivity-enhanced conditionally-replicative adenovirus ofclaim 1, wherein said early gene is conditionally regulated by meansselected from the group consisting of a tissue-specific promoteroperably linked to said early gene and a mutation in said early gene. 9.The infectivity-enhanced conditionally-replicative adenovirus of claim8, wherein said tissue-specific promoter is from a gene encoding aprotein selected from the group consisting of prostate specific antigen,carcinoembryonic antigen, secretory leukoprotease inhibitor,alpha-fetoprotein, vascular endothelial growth factor, CXCR4 andsurvivin.
 10. The infectivity-enhanced conditionally-replicativeadenovirus of claim 1, wherein said infectivity-enhancedconditionally-replicative adenovirus carries a therapeutic gene in itsgenome.
 11. The infectivity-enhanced conditionally-replicativeadenovirus of claim 10, wherein said therapeutic gene is a herpessimplex virus thymidine kinase gene.
 12. A method of killing tumor cellsin an individual, comprising the steps of: pretreating said individualwith an effective amount of the infectivity-enhancedconditionally-replicative adenovirus of claim 11; and administeringganciclovir to said individual.
 13. A method of providing adenoviralgene therapy in an individual, comprising the steps of: administering tosaid individual a therapeutic dose of an infectivity-enhancedconditionally-replicative adenovirus, wherein said adenovirus possessesenhanced infectivity towards a specific cell type due to modification orreplacement of the fiber of a wildtype adenovirus, wherein saidmodification or replacement results in enhanced infectivity relative tosaid wildtype adenovirus, and wherein said infectivity-enhancedconditionally-replicative adenovirus has at least one conditionallyregulated early gene, said early gene conditionally regulated such thatreplication of said infectivity-enhanced conditionally-replicativeadenovirus is limited to said specific cell type.
 14. The method ofclaim 13, wherein said administration is by means selected from thegroup consisting of intravenously, intraperitoneally, systemically,orally and intratumorally.
 15. The method of claim 13, wherein saidindividual has cancer.
 16. The method of claim 13, wherein said cell isa tumor cell.
 17. The method of claim 13, wherein said modification orreplacement to the fiber results in coxsackie-adenovirusreceptor-independent gene transfer with respect to the type 5 receptor.18. The method of claim 13, wherein said modification or replacement tothe fiber is selected from the group consisting of introducing a ligandinto the HI loop of said fiber, replacing said fiber with a substituteprotein which presents a targeting ligand, and introducing a fiber knobdomain from a different subtype of adenovirus.
 19. The method of claim18, wherein said ligand is selected from the group consisting ofphysiological ligands, anti-receptor antibodies and cell-specificpeptides.
 20. The method of claim 18, wherein said ligand comprises atripeptide having the sequence Arg-Gly-Asp (RGD).
 21. The method ofclaim 18, wherein said ligand comprises a peptide having the sequenceCDCRGDCFC.
 22. The method of claim 13, wherein said early gene isconditionally regulated by means selected from the group consisting of atissue-specific promoter operably linked to said early gene and amutation in said early gene.
 23. The method of claim 22, wherein saidtissue-specific promoter is from a gene encoding a protein selected fromthe group consisting of prostate specific antigen, carcinoembryonicantigen, secretory leukoprotease inhibitor, alpha-fetoprotein, vascularendothelial growth factor, CXCR4 and survivin.
 24. The method of claim13, wherein said adenovirus carries in its genome a therapeutic gene.